Isoforms of 2,4-dienoyl-CoA reductase (EC 1.3.1.34), which is the key enzyme in the beta-oxidation of fatty acids with double bonds, have been studied in rat heart and liver. Electrofocusing and adsorption chromatography on hydroxyapatite were used to separate the reductase activity in tissue homogenates into two peaks, one peak in each experiment being identified as the previously purified mitochondrial reductase. The novel activity was partially purified from rat liver by means of ammonium sulphate precipitation, anion-exchange chromatography on DEAE-cellulose (DE-52), hydrophobic chromatography on Phenyl-Sepharose and dye-ligand binding chromatography (Blue Sepharose). Taking into account the contribution of the different reductases to the total activity in rat liver, the overall purification for the novel isoform was 1900-fold. Ultracentrifugation on a sucrose gradient gave an M(r) of 50,000 and size-exclusion chromatography on Superdex 200 an M(r) of 60,000. The antibody against the previously characterised reductase did not cross-react with this novel isoform, but the distribution of the activity peaks in heart and liver tissue, and an electrofocusing experiment with isolated mitochondria, both pointed to a mitochondrial origin. The novel reductase was estimated to account for 80% (50%) of the total reductase activity in rat heart (liver) homogenate measured with 2,4-hexadienoyl-CoA. The present results, together with those previously published, suggest that mammals have at least three reductase isoforms: two in mitochondria and a third one in peroxisomes, but the peroxisomal activity has not been characterised so far.