The purpose of this study was to determine whether people could naturally produce urine sufficiently dilute to meet the federal criteria for a “substituted” specimen. The United States Department of Transportation Regulations (49 Code of Federal Regulations Part 40) defines a urine specimen as substituted if it has a creatinine concentration of ≤5 mg/dL and a specific gravity of ≤1.001 or ≥1.020. These criteria have been criticized based on the contention that an insufficient number of specimens had been tested from the same urine sample for both creatinine and specific gravity measurements. We reviewed the results of 803,130 random urine specimens measured for creatinine and/or specific gravity in a hospital-based laboratory. In this database, 13,467 urine specimens had both creatinine and specific gravity measurements. None of these 13,467 paired urine specimens met the lower limit of specific gravity (≤1.001) and creatinine (≤5 mg/dL) criteria for a Department of Transportation substituted specimen. We also examined the medical records of those patients meeting even one of the two criteria; creatinine concentration ≤5 mg/dL or specific gravity ≤1.001. These patients were neonatal, moribund, or so severely ill that essentially none could have been among the working population. These data in patients with various pathologic states support our belief that normal individuals do not produce urine dilute enough to meet the lower limit of the specific gravity (≤1.001) and creatinine (≤5 mg/dL) required for meeting substituted specimen criteria. Eleven patients met the criteria for a substituted specimen, with elevated specific gravity of ≥1.020 and creatinine concentration of ≤5 mg/dL; however, these patients were seriously ill or terminally ill.