The inhibitory effect of fluconazole (FCZ), a bis-triazole antimycotic, on mouse hepatic microsomal cytochrome P-450-mediated drug-metabolizing enzyme system was compared with those of ketoconazole (KCZ) in vivo and in vitro. Additionally, the change in the hepatic oxidative drug-metabolizing capacity in humans treated with FCZ was followed. The pentobarbital sleeping time in mice given a single dose of 1-10 mg/kg of FCZ or 30-50 mg/kg of KCZ was prolonged significantly, and the potency of FCZ for the prolongation of sleeping time was greater than that of KCZ. In contrast, in vitro the affinity and the inhibitory potency of FCZ for cytochrome P-450 and aminopyrine N-demethylation were 4- to 6-fold smaller than those of KCZ. However, the order of the inhibitory potencies among antimycotics for this enzyme system in vitro was reversed by the addition of albumin into the reaction mixture. These results indicate that the difference in the plasma protein binding properties between FCZ and KCZ is an important factor which leads to a reverse in the order of their inhibitory potencies for this enzyme system in vitro and in vivo. The ratio of 6β-hydroxycortisol (6β-OHF) to cortisol (F) in urine, used as an indicator of oxidative drug-metabolizing capacity in humans, decreased to 50% of the original level during treatment with 200 mg/d of FCZ. These findings indicate that the inhibitory effect of FCZ on hepatic microsomal cytochrome P-450-mediated drug-metabolism in vivo was unexpectedly potent from its potential obtained in vitro experiments in the absence of albumin, and that FCZ could be a potent inhibitor for hepatic oxidative drug-metabolism in humans.