Cortisol is metabolized to 6 beta-hydroxycortisol by the human cytochrome P-450 3A4, an enzyme implicated in the critical epoxidation reactions of aflatoxin B1 and certain polycyclic aromatic hydrocarbons. A method has been developed to measure the 6 beta-hydroxycortisol:cortisol ratio in human urine by reverse-phase high-performance liquid chromatography using diode-array detection. This method permits measurement of both cortisols in a single chromatographic run and has the potential to measure other metabolites useful for the determination of other human cytochrome P-450 activities. Using the 6 beta-hydroxycortisol:cortisol ratio as a marker of cytochrome P-450 3A4 activity adjusts for the circadian fluctuation in cortisol levels, providing a determination that is consistent over a 24-h period, obviating the need for 24-h urine collections. This high-performance liquid chromatography technique was used for the analysis of 14 different human urines and a range of ratios of 1.6 to 21.7 (mean +/- SE, 6.2 +/- 1.6) was found. These values are consistent with those reported in the literature. This method should be useful in molecular epidemiological studies investigating relationships between the susceptibility from environmental carcinogen exposure and cancer risk.