Peroxisomes in human hepatoblastoma cell line HepG2 have been studied using immunocytochemical, ultrastructural and biochemical techniques. By immunofluorescence, small spherical peroxisomes were seen next to rod-shaped and elongated forms. By electron microscopy and catalase cytochemistry, small particles with a diameter of 90 to 100 nm were found next to larger ones measuring up to 300 nm and some exhibiting tail-like extensions. Both the intensity of DAB-staining and the immunolabeling density for catalase were heterogenous in different peroxisomes. Contrary to a recent biochemical study, the enzyme alanine-glyoxylate-aminotransferase was found by double immunofluorescence and immunoelectron microscopy to be localized exclusively in peroxisomes of HepG2 cells. By Metrizamide density gradient centrifugation two populations of peroxisomes were isolated: a regular fraction banding at 1.20 g/cm3 with a mean diameter of 222 nm and a lighter peroxisome fraction banding at 1.14 g/cm3. The ratio of lipid beta-oxidation enzymes to catalase was significantly higher in peroxisomes with lower density than in the regular ones. These observations show clearly the existence of heterogenous populations of peroxisomes in HepG2 cells which may provide a useful model system for the investigation of biogenesis and metabolism of peroxisomes of human origin.