The activities and amounts of enzyme proteins of peroxisomal beta-oxidation in Japanese children with Zellweger syndrome were investigated. Cyanide-insensitive fatty acid oxidation, peroxisomal enoyl-CoA hydratase and 3-oxoacyl-CoA thiolase activities were not detectable in liver tissue at autopsy, whereas the activities of mitochondrial enoyl-CoA hydratase, 3-oxoacyl-CoA thiolase and carnitine palmitoyltransferase were similar to those in the healthy controls. On immunoblot analysis, immunoreactive proteins of peroxisomal acyl-CoA oxidase, bifunctional protein and 3-oxoacyl-CoA thiolase were not detected in the livers, kidneys and fibroblasts from the patients. Proteins of catalase and some enzymes of mitochondrial fatty acid oxidation were similar as in normal controls. These data indicate that increased levels of very-long-chain fatty acids in Zellweger syndrome are due to the lack of the enzyme proteins of peroxisomal beta-oxidation.