Quick freezing of tissues, with freeze-substitution fixation, not only provides superior ultrastructural detail in cells and tissues but also retains soluble substances within their normal intracellular compartments. Combining this procedure with postembedding protein A-gold immunocytochemical technique permits precise localization of antigenic sites at the electron microscopic level.Biochemical and cytochemical studies have demonstrated variously that xanthine oxidase, one of the flavoprotein enzymes involved in urate metabolism, is localized in cytozol, peroxisomal membrane, and/or peroxisomal crystalline cores of rat hepatocytes. This study is designed to determine the localization of xanthine oxidase in rat hepatocyte by high resolution immuno-electron microscopy.Adult Wistar rats, 150-230 g, maintained on standard rat food pellets and water ad libitum were fasted for 12 h before sacrifice. To examine the presence of xanthine oxidase in peroxisomes in rat hepatocyte, some animals were treated with intramuscular injections of 250 mg (per day) of clofibrate, a hypolipidemic drug, for 10 days to generate peroxisome proliferation. Fresh tissue blocks were quickly frozen by the metal contact method, using liquid helium, and then freeze-substituted with either 2% OsO4 in acetone or 0.3% glutaraldehyde-acetone solution.