The role of several P450 enzymes in the metabolism of diazepam (DZ) has been investigated. Hepatic microsomes of adult male rats were pretreated with antisera raised against the P450s CYP3A2, 2B1, 2C6, 2C11, 2D1 and 2E1, and their influence on the subsequent metabolism of DZ was determined by simultaneously measuring the changes in the relative rates of formation of its metabolites. Several forms of P450 were found to be positively involved in DZ metabolism. Antisera of the "male-specific" P450 enzyme CYP2C11 partially inhibited both DZ N-demethylase and C3 hydroxylase activities (60%) which resulted in decreased formations of N-desmethyl-DZ (NDZ) and 3-hydroxy-DZ (3HDZ), respectively. In a reconstitution experiment with the purified enzyme, CYP2C11 predominantly catalysed the formation of NDZ from DZ. Antisera of a further male-specific P450 CYP3A2 strongly inhibited (95%) the C3 hydroxylase of DZ and thus 3HDZ formation. A corresponding reconstitution experiment with this same P450 enzyme gave 3HDZ as principal product. CYP2D1 antisera inhibited the aromatic hydroxylation of DZ (98%) and subsequent formation of 4'-hydroxy-DZ (4'HDZ). This enzyme was also observed to inhibit DZ N-demethylase activity (60%). A reconstitution experiment with pure CYP2D1 catalysed the formation of both 4'HDZ and NDZ.