We searched for novel Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by an improved method using peroxisome targeting sequence (PTS) of Pex3p (amino acid residues 1–40)-fused enhanced green fluorescent protein (EGFP). From mutagenized TKaEG3(1–40) cells, the wild-type CHO-K1 stably expressing rat Pex2p and of rat Pex3p(1–40)-EGFP, numerous cell colonies resistant to the 9-(10-pyrene) nonanol/ultraviolet treatment were grown. These colonies were examined for intracellular location of Pex3p(1–40)-EGFP. By this method, we have isolated one CHO cell mutant, ZPEG403, which was found to belong to complementation group G (CG-G). Expression of the human peroxin, Pex3p cDNA encoding a 373-amino-acid peroxisomal membrane protein morphologically and biochemically restored peroxisome biogenesis, including peroxisomal membrane assembly, in ZPEG403 cells. Mutation and genomic DNA PCR analyses showed that, the dysfunction of Pex3p in ZPEG403, was due to one base (A) substitution in place of (G) in the first base of splicing site at the boundary of exon 6 and intron 6 of PEX3 gene, giving rise to remaining of all of intron 6, thereby inducing 81 bp insertion between positions 523–524 of PEX3 ORF, resulting in deletion of 200 amino acid residues from the C-terminus of Pex3p and a frame shift inducing both 18-amino-acid substitution and an early termination codon.