Urinary 6 beta-hydroxycortisol (6 beta OHF) is a specific marker of the induction of the cytochrome P-450 IIIA. Often, the production of 6 beta OHF is expressed from 24-h urine collection as the ratio 6 beta OHF/17-hydroxycorticosteroids or even better 6 beta OHF/free cortisol in order to adjust for minor day to day variation in adrenal cortisol production. A 24-h urine collection is a non-invasive method applicable to clinical studies in adult man, but more difficult to realize in newborn and infants. Therefore, we have tested the validity of a single urine sampling for the determination of the 6 beta OHF/free cortisol ratio as a clinical test to estimate cytochrome P-450 IIIA enzyme activity. Urinary 6 beta OHF and free cortisol have been measured in 19 healthy volunteers by high performance liquid chromatography methods using the same extraction procedure. A good correlation was found between 6 beta OHF/free cortisol ratio in morning urine samples and in 24h-collections (r = 0.899). Thus, a morning spot urine sampling is a simple procedure that may be helpful for investigations on cytochrome P-450 IIIA enzyme activity in humans, particularly in newborn and in children.