4-Hydroxylation is an important pathway of tamoxifen metabolism because the product of this reaction is intrinsically 100 times more potent as an oestrogen receptor antagonist than is the parent drug. Although tamoxifen 4-hydroxylation is catalysed by human cytochrome P450 (CYP), data conflict on the specific isoforms responsible. The aim of this study was to define unequivocally the role of individual CYPs in the 4-hydroxylation of tamoxifen by human liver microsomes. Microsomes from each of 10 human livers catalysed the reaction [range = 0.6-2.9 pmol/mg protein/min (1 microM substrate concentration) and 6-25 pmol/mg protein/min (18 microM)]. Three of the livers with the lowest tamoxifen 4-hydroxylation activity were from genetically poor metabolisers with respect to CYP2D6. Inhibition of activity by quinidine (1 microM), sulphaphenazole (20 microM) and ketoconazole (2 microM), selective inhibitors of CYPs 2D6, 2C9 and 3A4, respectively, was 0-80%, 0-80% and 12-57%. The proportion of activity inhibited by quinidine correlated positively with total microsomal tamoxifen 4-hydroxylation activity (rs = 0.89, P < 0.01), indicating a major involvement of CYP2D6 in this reaction. Recombinant human CYPs 2D6, 2C9 and 3A4 but not CYPs 1A1, 1A2, 2C19 and 2E1 displayed significant 4-hydroxylation activity. Similar inhibition and correlation experiments confirmed that tamoxifen N-demethylation is catalysed predominantly by CYP3A4. These findings indicate that the 4-hydroxylation of tamoxifen is catalysed almost exclusively by CYPs 2D6, 2C9 and 3A4 in human liver microsomes. However, the marked between-subject variation in the contribution of these isoforms underlines the need to study metabolic reactions in a sufficient number of livers that are characterised with respect to a range of cytochrome P450 activities.