It has been suggested that cortisol 6 beta-hydroxylase activity specifically reflects cytochrome P4503A (CYP3A) levels in the liver. However, we have previously reported that the metabolism of cortisol in human liver fractions in vitro is extremely complex and variable, and therefore complete metabolite analysis must be undertaken if 6 beta-hydroxycortisol is to be used as a marker of CYP3A activity. In the present study, the metabolism of [3H]cortisol by hepatic microsomes from various animal species, and by cytosol from male and female rats, has been defined and compared with metabolites formed by human liver microsomes. Metabolites were characterized by co-chromatography with authentic standards, mass spectrometry, and quantified by radiometric HPLC. The results show that all microsomes prepared from animal species studied (male and female rat, male and female guinea-pig, male hamsters and mice) can metabolize cortisol, although the metabolic profiles are both quantitatively and qualitatively different from that obtained with human microsomes. In general the metabolic profiles for animal microsomes are simpler: hamster, mouse and guinea pig show only 6 beta-hydroxylase and 11 beta-dehydrogenase activity, although male rat shows both of these and 20 beta-reductase activity while the female rat possesses all of the above as well as the ability to reduce the A-ring (delta 4-reductase and 3-oxidoreductase activities). The female rat also produces two metabolites undetected in humans. Incubations with male rat cytosol generated 20 beta-dihydrocortisone as the major metabolite, and several unidentified minor polar metabolites, whereas female cytosolic products were identical to those generated by human cytosol, the major metabolite being 3 alpha,5 beta-tetrahydrocortisol. In conclusion, our studies have shown that hepatic cortisol metabolism is extremely variable amongst the species investigated and that the hamster provides the simplest model with which to explore cortisol 6 beta-hydroxylase activity.