cDNA clones of rat peroxisomal 3-ketoacyl-CoA thiolase were isolated. By blotting analysis using the cDNAs as probes, the mRNA for this enzyme was estimated to be about 1.9-kilobase pairs. Elevation of mRNA levels in the liver with administration of di(2-ethylhexyl)phthalate was also evident. Sequencing analysis revealed 1,272 bases of the open reading frame which encoded 424 amino acid residues. Amino acid sequence data on six tryptic peptides and the amino terminus of the purified enzyme confirmed the cDNA sequence. The precursor of peroxisomal thiolase contains at its amino terminus a peptide extension of 26 residues. The mature enzyme is composed of 398 amino acids and the molecular weight is 41,074. The presequence has a net positive charge, lacks a long stretch of hydrophobic residues, and contains a cluster of serine residues. When the primary structure of the precursor was compared to structure of known peroxisomal proteins, there was no common homologous sequence. Peroxisomal thiolase exhibits a significant sequence homology with the mitochondrial thiolase. Possible location of the transport signal of the peroxisomal thiolase is discussed. Acyl-CoA binding sites were also located on primary structures of the two thiolases. The occurrence of interrupting sequences in several clones likely originates from intron sequences.