The impact of ketoconazole (200 mg for 7 days) on the kinetics of oral prednisone and intravenous prednisolone and on the apparent activity of the 6 beta-hydroxylase was investigated in 10 healthy volunteers. The ratio of urinary 6 beta-OH-cortisol/17-OH-corticosteroids declined by greater than 50% and the urinary excretion of 6 beta-OH-prednisolone decreased more than twofold in all subjects. The decline of the activity of the 6 beta-hydroxylase was associated with impaired metabolic and renal clearances of total and unbound prednisolone. The ratios of the AUCs of prednisolone/prednisone after oral prednisone and intravenous prednisolone were independent of the administration of ketoconazole, suggesting that the enzymes responsible for the interconversion of prednisolone in equilibrium prednisone were not affected by ketoconazole. Thus ketoconazole inhibits 6 beta-hydroxylase and increases the exposure of the body to the biologically active unbound prednisolone after oral prednisone or intravenous prednisolone.