To elucidate structural relationships between the mitochondrial and peroxisomal isozymes of beta-oxidation systems, cDNA of the mitochondrial enoyl-CoA hydratase was cloned and sequenced. The 1454-bp cDNA sequence contained a 870 bp of open reading frame, encoding a polypeptide of 290 amino acid residues. When compared with the amino-terminal sequence of the mature enzyme, the predicted sequence contained a 29-residue presequence at the amino terminus. This presequence had characteristics typical of a mitochondrial signal peptide. The primary structure of this enzyme showed significant similarity with the amino-terminal portion of sequence of the peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The carboxy-terminal part of the latter enzyme has sequence similarity with mitochondrial 3-hydroxyacyl-CoA dehydrogenase [Ishii, N., Hijikata, M., Osumi, T. & Hashimoto, T. (1987) J. Biol. Chem. 262, 8144-8150]. These findings suggest that the peroxisomal bifunctional enzyme has the hydratase and dehydrogenase functions on the amino- and carboxy-terminal sides, respectively. The mitochondrial beta-oxidation enzymes and the peroxisomal bifunctional enzyme may have common evolutionary origins.