1. Urinary excretion of 6 beta‐hydroxycortisol, hepatic microsomal cortisol 6 beta‐hydroxylase and the specific content of several forms of cytochrome P450 were measured in 8 to 14 patients before and after treatment with rifampicin (600 mg orally per day for 4 days). 2. Rifampicin treatment produced an average five fold increase in daily excretion of urinary 6 beta‐hydroxycortisol. 3. Cortisol 6 beta‐ hydroxylase activity increased from 15 +/‐ 6 pmol min‐1 mg‐1 in organ donors (considered as 'control subjects') to 87 +/‐ 31 pmol min‐1 mg‐1 in rifampicin treated patients. 4. Among three forms of human P450 (P450IA, IIC and IIIA), (1), (2), measured by Western blots, only P450IIIA was significantly induced by the antibiotic. 5. Only antibodies against P450IIIA selectively inhibited cortisol 6 beta‐ hydroxylase in human liver microsomes. 6. Cortisol 6 beta‐hydroxylase was correlated with P450IIIA specific content. 7. The urinary level of 6 beta‐hydroxycortisol correlated with liver microsomal cortisol 6 beta‐ hydroxylase and P450IIIA specific content. 8. We conclude that P450IIIA is predominantly responsible for cortisol 6 beta‐hydroxylase activity in human liver microsomes and that urinary 6 beta‐hydroxycortisol is a marker of the induction of this cytochrome P450.