The immunocytochemical localization of catalase and three enzymes of the peroxisomal lipid beta-oxidation system--acyl-CoA oxidase, the bifunctional protein enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase--in human liver biopsies was investigated by means of light and electron microscopy. The antisera raised against all four enzymes from rat liver cross-reacted with the corresponding proteins in homogenates of human liver as revealed by immunoblotting. For light-microscopic localization in glutaraldehyde-fixed Epon-embedded material, the removal of resin and controlled digestion with trypsin was necessary. At the ultrastructural level specific labeling for all four antigens was found by the protein A-gold technique in peroxisomes of liver parenchymal cells fixed with formaldehyde-low glutaraldehyde concentrations and embedded in Lowicryl K4M. In biopsies fixed with glutaraldehyde and embedded in Epon, treatment with metaperiodate or etching with sodium ethoxide improved the immunolabeling. After such treatment catalase showed the most intense labeling and acyl-CoA oxidase the weakest, the two other proteins exhibiting an intermediate immunoreaction. In material postfixed with osmium only catalase could be visualized in peroxisomes. The immunocytochemical investigation of peroxisomal proteins in human liver biopsies provides a simple and highly promising approach for further elucidation of the pathophysiology of peroxisomal disorders.