Metabolism of diazepam (DZP) was studied in vitro to clarify the involvement of different forms of hepatic cytochrome P450 (CYP) in rats, and humans of Japanese and Caucasian origin. Microsomal 3-hydroxylation was the major pathway of DZP metabolism in rats and was inhibited by anti-CYP3A antibodies. Purified CYP3As and CYP2C11 catalysed 3-hydroxylation and N-demethylation, respectively, in the reconstituted systems. The rates of both reactions in human liver microsomes depended on the substrate concentration: the rate of 3-hydroxylation was 3 ˜ 4 times higher than N-demethylation at 0.2 mM; the two activities were essentially the same at a lower substrate concentration (0.02 mM). Inhibitions of the N-demethylation by anti-CYP2C antibody and S-mephenytoin also depended on the substrate concentration and was detectable only at a low substrate concentration. Kinetic studies revealed the presence of two distinct catalytic activities for the N-demethylation; low Km and low Vmax, and high Km and high Vmax. The former activity seems to be mediated by a CYP2C P450 form. On the other hand, DZP 3-hydroxylation was rather selectively catalysed by a CYP3A P450 at the low and high substrate concentrations. These results were consistent with the observation in vivo that DZP N-demethylation and S-mephenytoin 4' -hydroxylation are closely correlated in humans. These results also suggest that the apparent discrepancy on the role of CYP forms in DZP metabolism in vitro and in vivo may reside in the difference in substrate concentration.