This study examined the cytochrome P450 (CYP) enzyme selectivity of in vitro bioactivation of lynestrenol to norethindrone and the further metabolism of norethindrone. Screening with well-established chemical inhibitors showed that the formation of norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC(50)=0.02 microM) and with CYP2C9 inhibitor sulphaphenazole (IC(50)=2.13 microM); the further biotransformation of norethindrone was strongly inhibited by ketoconazole (IC(50)=0.09 microM). Fluconazole modestly inhibited both lynestrenol bioactivation and norethindrone biotransformation. Lynestrenol bioactivation was mainly catalysed by recombinant human CYP2C9, CYP2C19 and CYP3A4; rCYP3A4 was responsible for the hydroxylation of norethindrone. A significant correlation was observed between norethindrone formation and tolbutamide hydroxylation, a CYP2C9-selective activity (r=0.63; p=0.01). Norethindrone hydroxylation correlated significantly with model reactions of CYP2C19 and CYP3A4. The greatest immunoinhibition of lynestrenol bioactivation was seen in incubations with CYP2C-Ab. The CYP3A4-Ab reduced norethindrone hydroxylation by 96%. Both lynestrenol and norethindrone were weak inhibitors of CYP2C9 (IC(50) of 32 microM and 46 microM for tolbutamide hydroxylation, respectively). In conclusion, CYP2C9, CYP2C19 and CYP3A4 are the primary cytochromes in the bioactivation of lynestrenol in vitro, while CYP3A4 catalyses the further metabolism of norethindrone.