49 CFR Part 40 Explained for DOT Collectors

Every DOT drug and alcohol test performed in the United States must follow one federal regulation: 49 CFR Part 40. Whether you’re a new collector learning the ropes or a working professional looking to sharpen your compliance knowledge, understanding Part 40 is non-negotiable. This article breaks it down in plain language — what the regulation covers, who it applies to, and exactly which sections every collector needs to know.

What Is 49 CFR Part 40?

Title 49 of the Code of Federal Regulations, Part 40 — commonly written as 49 CFR Part 40 or simply “Part 40” — is the federal regulation issued by the U.S. Department of Transportation (DOT) that standardizes drug and alcohol testing procedures across all DOT-regulated industries. It establishes a single, uniform testing framework that applies to trucking, aviation, railroad, transit, pipeline, and maritime employers nationwide.

Before Part 40 was enacted in 1994 (and significantly revised in 2001), each DOT operating agency — FMCSA, FAA, FRA, FTA, PHMSA, and USCG — had its own testing rules. The result was a patchwork of inconsistent procedures that created confusion for employers and service agents operating across multiple regulated sectors. Part 40 unified everything: one collection procedure, one form (the Federal Custody and Control Form), one laboratory standard, one Medical Review Officer process.

The regulation is administered by the DOT’s Office of Drug and Alcohol Policy and Compliance (ODAPC). It does not stand alone — it works in tandem with each agency’s own rules (e.g., FMCSA 49 CFR Part 382 for commercial motor vehicle drivers) that specify who must be tested and when. Part 40 governs how the testing is conducted.

Who Must Follow 49 CFR Part 40?

Part 40 applies to two broad groups: DOT-regulated employers and their service agents.

DOT-regulated employers are any company whose employees perform safety-sensitive functions under a DOT operating agency. This includes trucking companies (FMCSA), airlines and repair stations (FAA), railroads (FRA), transit agencies (FTA), pipeline operators (PHMSA), and maritime employers (USCG). If their employees drive commercial vehicles, fly aircraft, operate trains, or perform safety-critical work on regulated infrastructure, Part 40 applies.

Service agents are the network of trained professionals and organizations that carry out the actual testing on behalf of employers. Part 40 defines five categories of service agents:

• Collectors — Trained individuals who conduct urine specimen collections at collection sites. They are responsible for following the collection procedure, completing the CCF, and maintaining chain of custody.
• Medical Review Officers (MROs) — Licensed physicians who receive and review laboratory results, verify legitimate prescription explanations, and issue the final reported result to the employer.
• Substance Abuse Professionals (SAPs) — Licensed clinicians who evaluate employees who have violated DOT drug and alcohol regulations and recommend treatment or education before return-to-duty testing.
• HHS-certified laboratories — Laboratories certified by the Department of Health and Human Services (SAMHSA) that perform initial and confirmatory drug testing on DOT specimens. Only HHS-certified labs may test DOT specimens.
• Breath Alcohol Technicians (BATs) — Trained technicians who conduct breath alcohol tests using DOT-approved Evidential Breath Testing (EBT) devices.

Third Party Administrators (TPAs) and Consortia — the organizations that often coordinate testing programs for smaller employers — are also subject to Part 40 in their role as service agents.

Which Sections of 49 CFR Part 40 Do Collectors Need to Know?

Part 40 is organized into subparts — lettered sections that address different aspects of the testing process. Collectors are directly governed by four subparts:

What Does the Collection Procedure Under Part 40 Require?

Sections 40.61 through 40.73 contain the full collection procedure. Here’s what each stage requires:

1. 1

   Pre-collection steps (§ 40.61)

   The collector must ensure the collection site is secure and properly prepared: water sources turned off or blued, no unauthorized access, donor instructed to leave personal belongings outside. This step establishes the integrity of the environment before a specimen is provided.

2. 2

   Donor ID verification (§ 40.61(a))

   The collector must verify the donor’s identity using a government-issued photo ID (driver’s license, passport, or state ID) or, if unavailable, a positive identification by the employer’s representative. Collections without identity verification cannot proceed.

3. 3

   CCF initiation (§ 40.63)

   Before the donor provides a specimen, the collector initiates the Federal Custody and Control Form — completing Step 1 with the collection site and employer information, and checking the appropriate reason for test.

4. 4

   Specimen provision (§§ 40.65–40.67)

   The donor voids directly into the collection container provided by the collector. For standard (non-observed) collections, the restroom door is closed. The minimum required volume is 45 mL. If the donor cannot provide the minimum, the shy bladder protocol under § 40.193 is activated.

5. 5

   Temperature check and tamper inspection (§ 40.65)

   Within four minutes of receiving the specimen, the collector reads the temperature strip. Acceptable range: 90–100°F (32–38°C). Out-of-range temperature or any indication of substitution or adulteration requires the collector to direct an observed collection immediately and document the discrepancy on the CCF.

6. 6

   Specimen sealing (§ 40.67)

   The collector pours the specimen into the A and B bottles, applies tamper-evident seals with specimen ID labels, and the donor in the collector’s direct view initials both seals. Any break in mutual observation during this step requires the collection to be restarted.

7. 7

   CCF completion and donor copy (§§ 40.71–40.73)

   The collector completes Step 2 of the CCF (collection information and certification). The donor completes Step 5 (donor certification). The collector tears off and provides Copy 5 to the donor before the donor leaves. This copy is the donor’s record of the collection.

For a deep dive on the full chain-of-custody documentation process, including common errors and the complete 12-step collection sequence, see our dedicated guide.

What Are the Split Specimen Requirements Under 49 CFR Part 40?

Under 49 CFR Part 40, all DOT urine drug tests use a split specimen collection (also referred to as the A/B bottle method). Every collection produces two sealed specimens from the same void:

• Bottle A (primary specimen) — Minimum 30 mL. This is the bottle tested by the HHS-certified laboratory. If the initial immunoassay screen is positive, the lab performs GC/MS confirmatory testing on the same A bottle.
• Bottle B (split specimen) — Minimum 15 mL. This bottle is sealed, labeled, and held in reserve at the laboratory in frozen storage. It is only opened if the donor or the employer requests a split specimen retest to challenge the primary result.

The total minimum volume required for a split specimen collection is therefore 45 mL. If the donor provides less than 45 mL but more than 30 mL, only the A bottle is filled and the collection is documented as a non-split. If less than 30 mL is provided, the shy bladder protocol is initiated.

The split specimen system is one of the most important donor protections in Part 40. Before it was standardized, a donor challenging a positive result had no independent specimen available for retest. The A/B system ensures the donor always has recourse.

When Does 49 CFR Part 40 Not Apply?

Part 40 is mandatory for DOT-regulated testing — but not all workplace drug testing is DOT-regulated. Understanding the boundaries of the regulation prevents collectors from applying DOT procedures where they don’t belong (and vice versa).

Non-DOT testing — Testing performed by employers who are not DOT-regulated, or DOT-regulated employers testing employees who don’t perform safety-sensitive functions, is not governed by Part 40. These tests follow employer policy and state law, not federal procedure. The CCF is not used, and the collector is not required to follow the Part 40 collection sequence. A common mistake: using the DOT CCF for a non-DOT collection. This creates confusion and documentation problems — non-DOT tests must use the employer’s own non-federal form.

Reasonable suspicion testing — outside the Part 40 framework — While DOT agencies require reasonable suspicion testing for safety-sensitive employees, the employer’s decision to order the test (based on observed behavior) is governed by agency-specific rules, not Part 40. Part 40 governs the collection procedure once the test is ordered. The supervisor’s reasonable suspicion determination documentation is separate from, and not part of, the CCF.

State-regulated testing programs — Several states have their own drug and alcohol testing laws for non-regulated employers. These state programs may use different panels, different forms, and different procedures. Collectors who serve both DOT and non-DOT clients must be careful not to blend the two.

What Recent Amendments to 49 CFR Part 40 Should Collectors Know?

49 CFR Part 40 is a living regulation — the DOT updates it periodically as testing science evolves, new substances emerge, and program administration improves. Collectors are required to stay current with amendments; acting under outdated procedures is a compliance violation even if the collector was unaware of the change.

Key amendments in recent years include:

• Oral fluid testing authorization (2023) — The DOT finalized a rule allowing employers to use oral fluid (saliva) testing as an alternative to urine testing for DOT drug tests. Oral fluid collections follow a separate procedure; collectors must be specifically trained for this method.
• Drug panel expansions — The federal panel has been updated over time to add semi-synthetic opioids (oxycodone, oxymorphone, hydrocodone, hydromorphone) and revised cutoff levels for certain substances. Collectors don’t test specimens directly, but understanding what the panel covers helps you answer donor questions accurately.
• Remote collection site oversight updates — ODAPC has periodically updated guidance on the use of remote or mobile collection sites, employer-designated collection facilities, and collector qualification documentation.

The authoritative source for all current amendments, proposed rules, and ODAPC guidance is the DOT Office of Drug and Alcohol Policy and Compliance (ODAPC). Bookmark it. As a working collector, you should check ODAPC’s publications section at least annually — and any time you hear about a regulatory change in the field.

If you’re preparing for your initial certification, see our complete guide to becoming a certified DOT collector, including training requirements and what to expect on the proficiency demonstration. Once you’re certified, the DOT Collector Startup Checklist walks you through everything you need before your first collection.