The biogenesis, assembly and import of the peroxisomal enzyme catalase was studied in human skin fibroblasts from control persons and from patients with the Zellweger syndrome. For this purpose, two monoclonal antibodies were generated which are able to discriminate between the monomeric or dimeric form and the tetrameric, enzymically active conformation of the enzyme. Metabolic labelling studies showed that catalase is assembled to the tetrameric conformation within one hour after its synthesis, while it is still in the cytosol of the cell. Subsequently, the enzyme becomes particle-bound in the control cells, a process that is retarded by addition of the catalase inhibitor 3-amino-1,2,4-triazole. However, the tetramer remains in the cytosol in cells from Zellweger patients. It is concluded that newly synthesized catalase can be assembled to a tetramer in the cytosol in human skin fibroblasts. Unfolding of this tetramer prior to import into peroxisomes is indicated.