The in vivo disposition and in vitro metabolism of rifabutin, a new spiropiperidylrifamycin, were studied in rats and in microsomes from rat liver and enterocytes, respectively. After i.v. doses of 1,5, 10 and 25 mg/kg the systemic clearance was 0.7 to 1.0 liters/hr/kg; the volume of distribution was 4.4 liters/kg for the 1 mg/kg dose and 7.4 to 7.7 liters/kg for the 5 to 25 mg/kg doses, and the half-life ranged from 4.4 to 9.1 hr. Urinary and fecal excretion over 0 to 96 hr after i.v. administration of 25 mg/kg [14C]rifabutin accounted for 40.1 and 52.2% of the dose, respectively. Exteriorization of the bile duct showed that approximately 24% of the dose was eliminated in bile, > or = 98% as metabolites. Bioavailability after oral administration of 25 and 1 mg/kg rifabutin was > 90% and 44%, respectively, suggesting significant first-pass metabolism of the lower dose. Concentrations of rifabutin in gastric juice were 10 to 17 times higher than in blood, indicating extensive secretion into the stomach. Experiments with the isolated small intestinal loop demonstrated direct exsorption of the drug into the lumen. The rate of rifabutin metabolism by enterocyte microsomes was > 10 times higher than that by liver microsomes, i.e., 84 and 8 pmol/min/mg protein, respectively. Biotransformation of rifabutin in vivo and in vitro was markedly induced by dexamethasone and inhibited by erythromycin, suggesting that CYP3A is involved in the metabolism of rifabutin. Several metabolites, including 20-OH-rifabutin and 27-O-demethyl-rifabutin, isolated from urine and microsomes were identified by mass spectrometry and nuclear magnetic resonance spectroscopy.