Since Chinese hamster ovary (CHO) cells never express urate oxidase (UO), we tried to establish cell lines stably producing UO in order to elucidate the peroxisomal import process. The enzyme is a peroxisome targeting signal 1 (PTS1) protein harboring SKL motif at the carboxy-terminus [Biochem. Biophys. Res. Commun. 158 (1989) 991] and PEX5 protein (Pex5p) is supposed to be involved in the import process [Nat. Genet. 9 (1995) 115; J. Cell Biol. 130 (1995) 51]. We transfected a cDNA encoding rat UO into both wild type and PEX5-defective CHO cells to isolate each cell line stably producing the enzyme. While we examined the import process of UO in mutant cells, we noticed an interesting observation by using polyclonal antibody U1 or U2, which separately recognizes epitopes of UO. U1 antibody mainly interacts with epitopes in the amino-terminal region of UO. On the other hand, U2 antibody reacts with many epitopes distributed in the broad region of UO molecule. When UO produced in cultured cells was stained with U2 antibody, the enzyme was detected in peroxisomes of both wild type and PEX5-mutant cells. Whereas, U1 antibody stained the peroxisomal UO in wild type cells, but not in PEX5-mutant cells. These immunocytochemical observations suggest that the epitopes at the amino-terminal region of UO will be concealed in mutant cells. When the mutant cells were transfected with wild type PEX5 cDNA, U1 antibody came to react with UO in peroxisomes of mutant cells. The restoration indicates that the exposure of N-terminal epitopes of UO will depend upon the functional Pex5p. Immunoelectron microscopic observation showed that the peroxisomal import of UO was partially retarded in PEX5 mutant cells. The observation also supported the fact that UO was mainly localized in the peroxisomal matrix of wild type cells but in the membrane of mutant cells.