Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans approximately 5 kb, has 8 exons, and maps onto chromosome 7q34. The 5'-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes.