Biotransformation of rifabutin, an antibiotic used for treatment of tuberculosis in patients infected with the human immunodeficiency virus (HIV), and its interactions with some macrolide and antifungal agents were studied in human intestinal and liver microsomes. Both liver and enterocyte microsomes metabolized rifabutin to 25-O-deacetylrifabutin, 27-O-demethylrifabutin, and 20-, 31-, and 32-hydroxyrifabutin. The same products (except 25-O-deacetylrifabutin) were formed by microsomes from lymphoblastoid cells that contained expressed CYP3A4. The apparent Michaelis-Menten constant (Km); approximately 10 to 12 mumol/L) and maximal velocity (Vmax; approximately 100 pmol/min/mg of protein) values for CYP-mediated metabolism were similar in liver and enterocyte microsomes. Deacetylation of rifabutin (Km approximately 16 to 20 mumol/L and Vmax approximately 50 to 100 pmol/min/mg of protein) was catalyzed by microsomal cholinesterase. Clarithromycin, ketoconazole, and fluconazole inhibited CYP-mediated metabolism of rifabutin in enterocyte microsomes equally or more potently than in liver microsomes but had no effect on cholinesterase activity. Azithromycin did not inhibit in vitro metabolism of rifabutin. This study provides evidence that CYP3A4 and cholinesterase are major enzymes that biotransform rifabutin in humans and that intestinal CYP3A4 contributes significantly to rifabutin presystemic first-pass metabolism and drug interactions with macrolide and antifungal agents.