Delta(3)-Delta(2)-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme for the beta-oxidation of unsaturated fatty acids. The cDNA of the full-length rat liver Delta(3)-Delta(2)-enoyl-CoA isomerase was previously cloned as pAG847. PCR methodologies were used to subclone the gene encoding the functional Delta(3)-Delta(2)-enoyl-CoA isomerase from pAG847 with primers that were designed to add six continuous histidine codon to the 5(') primer. The PCR product was inserted into a pLM1 expression vector and overexpressed in Escherichia coli. The soluble expressed protein was purified with a nickel HiTrap chelating metal affinity column to apparent homogeneity based on Coomassie blue-stained SDS-PAGE and the molecular weight of the protein subunit was 30 kDa. The purified protein had a dimeric structure composed of identical subunits, and the molecular weight of the enzyme determined by gel chromatography was 60 kDa. Kinetic studies have been carried out and K(M) of 81 microM and V(max) of 292 micromol/min/mg were determined. The specific activity of the protein is 201 U/mg, which is significantly higher than that reported before for the same protein isolated from a natural source. The one-step purification of the highly active Delta(3)-Delta(2)-enoyl-CoA isomerase will greatly facilitate the further investigation of this enzyme through site-directed mutagenesis and enzyme catalyzed reactions with substrate analogues.