Simultaneous identification of optical isomers (D and L) of methamphetamine (MAMP), amphetamine (AMP), para(p)-hydroxy(OH)-MAMP, and para(p)-hydroxy(OH)-AMP in rat urine was attempted by high-performance liquid chromatography (HPLC). They were determined as benzoyl derivatives. After administration of D- or L-isomer (15 mg/kg), only D- or L-isomers of the above mentioned metabolites were found in rat urine. In rats administered racemic MAMP (15 mg/kg), each percent dose of L-isomers of MAMP or AMP excreted at four collection times up to 24 h was less than that of the D-isomer (L/D less than 1.00), but the doses of L-isomers of p-OH-MAMP and p-OH-AMP excreted were higher than those of D-isomers (L/D greater than 1.00). Simultaneous analysis within 36 minutes showed good peak resolution. The L/D ratio for each metabolite decreased with time. The total percent doses of D- or L-isomer excreted by 24 hours were about 50% of the administered dose, 23.70 +/- 1.45% for the D-isomer, 25.70 +/- 1.54% for the L-isomers. The total L/D ratio was 1.08 +/- 0.03. These results indicated that Wistar rats have no chiral isomerization enzyme of MAMP, and show stereoselective metabolism of DL-MAMP. In the optical isomer analysis of 11 MAMP powder samples and 28 human urine specimens obtained from Japanese abusers, only D-MAMP was detected in the powder, and D-MAMP and D-AMP were detected in urine, respectively. This suggested that humans have no chiral isomerization enzyme of D-MAMP.