The localization of urate oxidase in different tissues of rat and in the livers of selected mammalian species was investigated by immunoblot analysis and protein A-gold immunoelectron microscopy. Urate oxidase was purified from rat liver and used as an antigen to generate polyclonal antibodies in the rabbit. The antibodies were found to be monospecific by immunodiffusion and immunoblot analyses. By immunoblot analysis, urate oxidase was detected in the livers of rat, two strains of mice, hamster, dog, cat, and cow, but not in the Cynomolgus monkey and human liver. Urate oxidase was not detected by immunoblot method in rat kidney, jejunal mucosa, adrenal gland, testis, and pancreas. The subcellular localization of urate oxidase was ascertained by the protein A-gold immunocytochemical staining of the Lowicryl K4M embedded tissues. Urate oxidase was localized exclusively in the crystalloid core of the peroxisome in hepatic parenchymal cells of rat, mouse, hamster, dog, cat, and cow. The limiting membrane and the matrix of hepatic peroxisomes in these species were negative for the staining. The marginal plates of feline, canine, and bovine hepatic peroxisomes were also negative for urate oxidase. This enzyme was also not detected within the peroxisomes of human and monkey livers by the immunocytochemical technique. Peroxisomes (microperoxisomes) in extrahepatic rat tissues did not stain positively for urate oxidase by the protein A-gold immunocytochemical method, although they were positive for catalase. Fatty acyl-CoA oxidase was present in peroxisomes of jejunal mucosa, Leydig cells of test-is and pancreas but not in adrenal gland. Administration of a hepatic peroxisome proliferator, ciprofibrate or Wy-14643, failed to induce urate oxidase in rat liver. These results indicate that urate oxidase is a liver specific protein in rat and its localization within the liver peroxisomes of six mammals, excluding man and a nonhuman primate, and that its localization is limited exclusively to the crystalloid core. Unlike fatty acyl-CoA oxidase, urate oxidase does not appear to be inducible significantly by peroxisome proliferator treatment in the rat liver.