3-2trans-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme in mitochondrial beta-oxidation of unsaturated fatty acids in bacteria, plant and animal cells. The enzyme was isolated from rat liver mitochondria and purified to homogeneity by two chromatographic steps. Partial polypeptide sequences of the 29 kDa protein were derived from cyanogen bromide, tryptic, Lys-C, and protease V8 fragments by Edman degradation. Peptide-derived synthetic oligonucleotides were used for the isolation of a 990 bp long isomerase-specific cDNA from rat liver cDNA libraries. 867 bp encode the 289 amino-acid residues of the preisomerase with a molecular mass of 32,254 Da. The 1.3-kb mRNA is most strongly expressed in skeletal muscle followed by liver, heart, kidney, and weakly expressed in spleen and brain. In vitro transcription and translation yielded a 32 kDa polypeptide which was immunoprecipitated by anti rat isomerase antibodies. In the presence of mitochondria the 32 kDa precursor isomerase was processed during mitochondrial import to the 29 kDa mature form of the 3-2trans-enoyl-CoA isomerase with 264 amino-acid residues (Mr 29,706). A N-terminal signal sequence of 25 amino-acid residues directs the import into the mitochondrial matrix and is cleaved in two successive steps passing through an intermediate form of Mr 30,475. The two cysteine residues in positions 142 and 148 of the preisomerase are present as free thiol groups as shown by derivatization of the mature, native protein with the fluorescent label N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid. The mitochondrial 3-2trans enoyl-CoA isomerase shows significant homology and conserved amino-acid exchanges with the mitochondrial enoyl-CoA hydratase, the N-terminal domain of the bifunctional peroxisomal enoyl-CoA-hydratase:3-hydroxyacyl-CoA dehydrogenase and to extended domains of the alpha-subunit of the procaryotic beta-oxidation complex sharing enoyl-CoA isomerase, D(-)3-hydroxyacyl-CoA epimerase, enoyl-CoA hydratase and L(+)3-hydroxyacyl-CoA dehydrogenase activity, encoded by the fad B operon of E. coli.