1The metabolism of diazepam to its primary metabolites 3‐hydroxydiazepam (3HDZ) and nordiazepam (NDZ) was evaluated in human liver microsomes. The 3HDZ pathway was the major route of metabolism representing 90% of total metabolism with a Vmax /Kmratio of 0.50–7.26 μl min−1 mg −1 protein.2Inhibition of the two metabolic pathways of diazepam by omeprazole was investigated. The NDZ pathway was not affected by omeprazole whilst a Ki of 201±89 μm was obtained for the 3HDZ pathway (Km/Ki ratio of 3.0±0.9).3Inhibitory effects of omeprazole sulphone on the 3HDZ and NDZ pathways were also investigated. Omeprazole sulphone inhibited both pathways with similar Kis of 121±45 and 188±73 μm respectively (Km/Ki ratios of 5.2±2.3 and 3.3±1.5 respectively).4These in vitro data provide direct evidence for cytochrome P450 inhibition as the mechanism for the well documented diazepam‐omeprazole clinical interaction and indicate that omeprazole sulphone, as well as the parent drug, contribute to the inhibition effect.