The measurement of urinary 6 beta-hydroxycortisol (6 beta-OHF) has been widely used as a non-invasive clinical test to detect cytochrome P450 induction. Although only a minor biotransformation, 6 beta-OHF formation represents a sensitive target for many P450-inducing drugs and environmental chemicals in man. There is good evidence that an isozyme of the P450IIIA subfamily is predominantly responsible for 6 beta-hydroxylase activity and therefore it has been suggested that urinary 6 beta-OHF is a marker of the induction of P450IIIA. The basis of the present study was that in order to realistically assign to 6 beta-OHF the status of a P450IIIA marker we should characterize all the metabolites of cortisol produced by human liver and assess inter-liver variability. Incubations at 37 degrees C for 2 h contained [3H]cortisol (0.1 microCi, 1 or 50 microM), MgCl2 (10 mM), microsomal or cytosolic protein (3 mg), an NADPH-regenerating system and 1/15 M phosphate buffer (pH 7.4) to give a final volume of 0.5 ml. Extraction with ethyl acetate (2 x 2 ml) 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 microsomal incubations (n = 6 livers) produced 6 alpha-hydroxycortisol (6 alpha-OHF), 6 beta-OHF, 20 beta-dihydroxycortisol, 20 beta-dihydroxycortisone, cortisone, and 3 alpha, 5 beta-tetrahydrocortisone (3 alpha, 5 beta-THE), while five produced 6 beta-hydroxycortisone and four produced 3 alpha, 5 beta-tetrahydrocortisol (3 alpha, 5 beta-THF). The cytosolic incubations gave a much simpler metabolic profile, with 3 alpha, 5 beta-THF the major metabolite and 3 alpha, 5 beta-THE a minor metabolite. There was considerable inter-individual variability in metabolite profiles from microsomal incubations. 6 beta-OHF varied from 2.8 to 31.7%. Major metabolites were cortisone and 3 alpha, 5 beta-THE. Inter-liver variability was less for cytosolic incubations, the major metabolite always being 3 alpha, 5 beta-THF. In conclusion we have rigorously identified the hepatic metabolites of cortisol formed in vitro. The highly complex and variable hepatic metabolism of cortisol clearly limits the use of urinary 6 beta-OHF excretion as a marker of baseline P450IIIA activity in man.