Among the P450 forms tested, diazepam was significantly demethylated by CYP2B6, 2C9, 2C19, 3A4 and 3A5, with 2C19 exhibiting the highest rate at concentrations < 0.1 mM, and hydroxylated only by the latter three enzymes, with 3A5 being the most active. The N-demethylation activity of diazepam by 2C19 at a concentration of 20 μM was six times of that by 3A4. However, that by 2C9 was detected at only a trace level. CYP2C19, 3A4 and 3A5 of the ten human P450s catalysed the 3-hydroxylation of nordiazepam, and 2B6, the 2C subfamily and the 3A subfamily catalysed the N-demethylation of temazspam. CYP3A4 exhibited the highest activity of nordiazepam 3-hydroxylation and temazepam N-demethylation.