We report here the identification and characterization of human and mouse PECI, a novel gene that encodes a monofunctional peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase. Human and mouse PECI were identified on the basis of their sequence similarity to Eci1p, a recently characterized peroxisomal Delta(3),Delta(2)-enoyl-CoA isomerase from the yeast Saccharomyces cerevisiae. Cloning and sequencing of the human PECI cDNA revealed the presence of a 1077-base pair open reading frame predicted to encode a 359-amino acid protein with a mass of 39.6 kDa. The corresponding mouse cDNA contains a 1074-base pair open reading frame that encodes a 358-amino acid-long protein with a deduced mass of 39.4 kDa. Northern blot analysis demonstrated human PECI mRNA is expressed in all tissues. A bacterially expressed form of human PECI catalyzed the isomerization of 3-cis-octenoyl-CoA to 2-trans-octenoyl-CoA with a specific activity of 27 units/mg of protein. The human and mouse PECI proteins contain type-1 peroxisomal targeting signals, and human PECI was localized to peroxisomes by both subcellular fractionation and immunofluorescence microscopy techniques. The potential roles for this monofunctional Delta(3),Delta(2)-enoyl-CoA isomerase in peroxisomal metabolism are discussed.