The metabolism of the progestogen gestodene has been studied in human liver cytosol and microsomal incubations. Extraction with diethyl ether was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the cytosolic incubations (n = 4 livers) produced dihydrogestodene as the major metabolite, with lesser amounts of a tetrahydro derivative. It was not possible to separate the 5 alpha- and 5 beta-isomers of dihydrogestodene on the chromatographic system used. Values of Km and V(max) for the delta 4 reductase were determined. Androstenedione (Ki = 2.85 +/- 1.5 microM; n = 4) and cortisol (ki = 24.1 +/- 8.9 microM; n = 4) both inhibited the delta 4-reductase. In contrast desogestrel showed virtually no inhibition at concentrations up to 200 microM. The major microsomal metabolite of gestodene was a hydroxylated derivative although mass spectral analysis was unable to determine the position of insertion of the hydroxyl moiety. The hydroxylation of gestodene (1 microM) was markedly inhibited by ketoconazole (IC50 < 0.1 microM), and also by cyclosporin. This suggests that the cytochrome P450 isozyme CYP3A4 is important in gestodene metabolism. Theophylline and tolbutamide (substrates of CYPIA and CYP2C, respectively) did not affect gestodene metabolism at concentrations up to 100 microM. In conclusion, the major biotransformation of gestodene (A-ring reduction) occurs in the cytosolic fraction of human liver. Microsomal hydroxylation appears to be catalysed by CYP3A4.