The ratio of the hydrophilic metabolite 6β-hydroxycortisol to its parent compound cortisol has recently been demonstrated to be a specific marker for human CYP3A oxygenase activity. We have developed a sensitive and simple single-run high-performance liquid chromatographic method for the quantification of urinary free cortisol and 6β-hydroxycortisol using dexamethasone as internal standard. The urine samples (1 ml) are applied to Sep-Pak cartridges, which are washed with water and eluted with ethyl acetate-diethyl ether (4:1, v/v). The organic extracts are washed sequentially with alkaline and acidic solutions saturated with sodium sulfate and subsequently concentrated to dryness. After reconstitution in ethanolic water, the samples are analyzed on a reversed-phase gradient system using ultraviolet absorbance detection at 254 nm. The within- and between-day coefficients of variation (C.V.) for the assay where both in the range of 5-10%. The reference interval for the 6β-hydroxycortisol/cortisol ratio of eleven healthy non-smoking subjects was 2.77-26.88 with an average of 10.09 ± 6.89 (S.D.). The method constitutes an improvement over previous methods and is suitable for routine assessment of the 6β-hydroxycortisol/cortisol ratio requiring only 1 ml of urine or less.