Ketoconazole, an antifungal agent and inhibitor of certain mammalian cytochrome P-450-dependent enzymes, was studied for its effects on the in vitro and in vivo metabolism of all-trans-retinoic acid (RA). In vitro, ketoconazole (Ki = 0.75 microM) inhibited, in an apparently competitive manner, the cytochrome P-450-mediated metabolism to 4-hydroxy- and 4-keto-retinoic acids by hamster liver microsomes. In vivo, ketoconazole suppressed the formation of polar RA metabolites by normal rats dosed intrajugularly with 200 ng of [3H]RA. After p.o. treatment with ketoconazole (2.5-40 mg/kg) given 1 hr before the [3H]RA injection, the radioactivity extracted from the liver consisted of 25 to 50% polar metabolites (control 66 +/- 1%) and 50 to 75% undegraded RA (control 34 +/- 1%) as evidenced by reverse-phase high-performance liquid chromatography. Time course experiments showed that ketoconazole's inhibitory effects lasted for 3 hr. Our data indicate the quantitative importance of the cytochrome P-450 enzymatic pathway in the biotransformation of RA. They also suggest that ketoconazole is capable of prolonging the biological half-life of RA and of improving the tissue levels of this compound.