We isolated and analyzed the genes for rat peroxisomal 3-ketoacyl-CoA thiolase. Two active genes (termed A and B), both likely to be coding for the peroxisomal thiolase, were identified per haploid rat genome. These genes span approximately 9 and 11 kilobases, respectively, and both contain 12 exons and 11 introns, with overall similar organizations. The nucleotide sequences are highly conserved in the coding regions and in many introns. The A and B genes code for slightly different amino acid sequences, and the known cDNA sequence (Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8151-8158) corresponds to the B gene product. By in vitro transcription/translation, the A gene was shown to produce a thiolase precursor having a 10-residue longer amino-terminal presequence than that of the B gene product. Primer extension analysis revealed that the A gene is constitutively expressed and is not influenced by the administration of a peroxisome proliferator, whereas the B gene is markedly activated by the same treatment. Several common sequences were noted in the 5'-flanking regions of the B gene and in the genes of two other peroxisomal beta-oxidation enzymes, induced in parallel by peroxisome proliferators.