We have reported previously that the formation of a 3',4'-dihydroxylated metabolite of phenytoin (3',4'-diHPPH) by human liver microsomal cytochrome P450 (P450) is enhanced by the addition of human liver cytosol [Komatsu et al., Drug Metab Dispos 2000;28:1361-8]. The enhancing factor was determined in this study. The addition of cytosolic proteins precipitated by 50% ammonium sulfate to incubation mixtures increased the rate of microsomal 3',4'-diHPPH formation. This fraction was separated further by diethylaminoethyl-, carboxymethyl-, and hydroxyapatite-column chromatography. The amino acid sequence of the purified protein of approximately 55kDa by electrophoresis revealed this protein to be a catalase. The addition of purified or authentic catalase to the incubation mixtures increased the rates of microsomal 3',4'-diHPPH formation from 3'- and 4'-hydroxylated metabolites and from phenytoin in a concentration-dependent manner. In reconstituted systems containing CYP2C9, CYP2C19, and CYP3A4, the formation of 3',4'-diHPPH was also enhanced by catalase to different extents. This is the first report that catalase in livers enhances drug oxidation activities catalyzed by P450 in human liver microsomes.