The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3 alpha,5 beta-tetrahydrocortisol; 3 alpha,5 beta-THF), while microsomal incubations generate upto 7 metabolites, including 6 beta-hydroxycortisol (6 beta-OHF), and 6 beta-hydroxycortisone (6 beta-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [3H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The Km value for 6 beta-OHF and 6 beta-OHE formation was 15.2 +/- 2.1 microM (mean +/- SD; n = 4) and the Vmax value 6.43 +/- 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6 beta-hydroxylase was ketoconazole (Ki = 0.9 +/- 0.4 microM; n = 4), followed by gestodene (Ki = 5.6 +/- 0.6 microM) and cyclosporine (Ki = 6.8 +/- 1.4 microM). Both betamethasone and dexamethasone produced some inhibition (Ki = 31.3 and 54.5 microM, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 +/- 11.2 microM (n = 4) and the Vmax value 107.7 +/- 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (Ki = 17.8 +/- 3.3 microM) and gestodene (Ki = 23.8 +/- 3.8 microM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.