KNI-272 is a tripeptide protease inhibitor for treating human immunodeficiency virus type 1 (HIV-1). In in vitro stability studies using rat tissue homogenates, KNI-272 concentrations in the liver, kidney, and brain decreased significantly with time. Moreover, in tissue distribution studies, KNI-272 distributed highly to the liver, kidney, and small intestine in vivo. From these results and reported physiological parameters such as the tissue volume and tissue blood flow rate, we considered the liver to be the main organ which takes part in the metabolic elimination of KNI-272. Then the hepatic metabolism of KNI-272 was more thoroughly investigated by using rat liver microsomes. KNI-272 was metabolized in the rat liver microsomes, and five metabolites were found. The initial metabolic rate constant (k_metabolism) tended to decrease when the KNI-272 concentration in microsomal suspensions increased. The calculated Michaelis-Menten constant (K_m) and the maximum velocity of KNI-272 metabolism (V_max), after correction for the unbound drug concentration, were 1.12 ± 0.09 µg/ml (1.68 ± 0.13 µM) and 0.372 ± 0.008 µg/mg of protein/min (0.558 ± 0.012 nmol/mg of protein per min), respectively. The metabolic clearance (CL_int,metabo), calculated as V_max/K_m, was 0.332 ml/mg of protein per min. Moreover, by using selective cytochrome P-450 inhibitors and recombinant human CYP3A4 fractions, KNI-272 was determined to be metabolized mainly by the CYP3A isoform. In addition, ketoconazole, a representative CYP3A inhibitor, inhibited KNI-272 metabolism competitively, and the inhibition constant (K_i) was 4.32 µM.