Seven healthy male volunteers received a single oral dose of 5 mg, 10 mg, or 20 mg of d-amphetamine. Urine was collected at 2, 4, 8, 12, 18, and 24 h post-dose. Total urine volume was measured, and pH and creatinine were determined. All specimens were analyzed by TDx Amphetamine/Methamphetamine II (TDx), Emit-d.a.u. Monoclonal Amphetamine/Methamphetamine (EM), and Emit II Amphetamine/Methamphetamine (EII) immunoassays at a cutoff value of 1000-ng/mL amphetamine. Quantitation of urinary amphetamine in all specimens was performed by gas chromatography-mass spectrometry. All urine testing results by the three immunoassays, EM, EII, and TDx, were in agreement; there were no discordant findings. Of the 42 total urine specimens collected following a 5-mg dose of amphetamine, only 8 (19%) screened positive by immunoassay. Twenty-four of 36 (67%) urine specimens yielded positive responses following a 10-mg dose, and 37 of 42 (88%) were positive by immunoassay following a 20-mg dose. These data demonstrate the present guideline for regulated forensic urine drug testing (FUDT) for amphetamine with a screening cutoff of 1000 ng/mL is too high to consistently detect the administration of a single 5-mg oral dose of d-amphetamine. There was considerable overlap of amphetamine concentrations in individual specimens following the various doses. Peak urinary amphetamine ranged from 620 to 3160 ng/mL following 5-mg doses. The time to peak concentration also varied widely at each dose, occurring in urines collected 2 to 18 h post-administration. The mean percent of dose excreted as unchanged amphetamine over 24 h at each dose ranged from 35 to 44%. The data demonstrated that amphetamine excretion increases with increasing urine flow and decreasing urine pH. Thus, a positive FUDT result for amphetamine means only that the individual was administered or self-administered amphetamine at some time prior to collection of the specimen.