Both ritonavir and indinavir were readily metabolized by human intestinal microsomes. Comparison of the patterns of metabolites in incubations with enterocyte microsomes and expressed cytochrome P450 (CYP) isozymes and immunoinhibition and chemical inhibition studies showed the essential role of the CYP3A subfamily in the metabolism of both protease inhibitors by the small intestine. Ritonavir was similarly biotransformed by microsomes containing expressed CYP3A4 or CYP3A5 isozymes (K_M = 0.05–0.07 μM,V _max = 1–1.4 nmol/min/nmol CYP). In contrast, both the patterns of metabolites and the enzyme kinetic parameters for the metabolism of indinavir by expressed CYP3A5 (K_M = 0.21 μM,V _max = 0.24 nmol/min/nmol CYP) and CYP3A4 (K_M = 0.04 μM,V _max = 0.68 nmol/min/nmol CYP) were different. The biotransformation of both indinavir and ritonavir in human enterocyte microsomes was characterized by very lowK_M values (0.2–0.4 μM for indinavir and <0.1 μM for ritonavir). The V _maxfor indinavir metabolism was greater in enterocyte (163 ± 35 pmol/min/mg protein) than in liver (68 ± 44 pmol/min/mg protein) microsomes. The metabolism of ritonavir in liver and enterocyte microsomes was associated with inactivation of CYP3A. The initialV _max for ritonavir metabolism by enterocyte microsomes was 89 ± 59 pmol/min/mg protein. The apparent inactivation rate constants for intestinal CYP3A and expressed CYP3A4 were 0.078 and 0.135 min⁻¹, respectively. Metabolic inactivation of CYP3A by ritonavir explains the improved bioavailability and pharmacokinetics of ritonavir and the sustained elevation of blood levels of other, concomitantly administered, substrates of CYP3A.