The metabolism of the progestogen oral contraceptive desogestrel (Dg) has been studied in vitro using human liver microsomes. Metabolites have been separated using radiometric high performance liquid chromatography and identified by co-chromatography with authentic standards and by mass spectrometry. All the livers examined ( n = 6) were able to form 3-keto desogestrel as the main identifiable metabolite and also the presumed intermediates 3α-hydroxydesogestrel (3α-OHDg) and 3β-hydroxydesogestrel (3β-OHDg). In addition, a large polar heterogenous peak was evident on the radiochromatograms which did not co-chromatograph with any known metabolites of desogestrel. Inter-individual variability in metabolite formation was seen. A number of drugs were examined for their propensity to inhibit desogestrel metabolism. Primaquine was the most potent tested having an IC 50 value (inhibitory concentration reducing overall metabolite production by 50%) of 30 μM. Cimetidine, trilostane and levonorgestrel failed to inhibit at 250 μM. With 3α-OHDg as substrate, 3α-hydroxysteroid dehydrogenase (3α-HSDH) activity was 1.0 ± 0.3 nmol min −1 mg −1 protein which was five times greater than the activity of the 3β-HSDH towards 3β-OHDg. Miconazole was the most potent inhibitor tested having IC 50 values of 14 and 95 μM for 3α- and 3β-HSDH respectively. Surprisingly, trilostane was without inhibitory effect on either enzyme, which contrasts with other data involving 3β-HSDH in steroidogenic tissue. Our observations with trilostane may reflect tissue differences in the enzyme and/or differences in endogenous vs exogenous steroids (i.e. in the conversion of 3β-OHDg to 3-ketodesogestrel there is no requirement for isomerization). Kinetic parameters of 3α-HSDH were also determined.