The mitochondrial metabolism of 5-enoyl-CoAs, which are formed during the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of beta-oxidation. Metabolites were identified spectrophotometrically and by high performance liquid chromatography. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by medium-chain acyl-CoA dehydrogenase (EC 1.3.99.3) to 2-trans-5-cis-octadienoyl-CoA, which was isomerized to 3,5-octadienoyl-CoA either by mitochondrial delta 3,delta 2-enoyl-CoA isomerase (EC 5.3.3.8) or by peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA in the presence of soluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a delta 3,5,delta 2,4-dienoyl-CoA isomerase. Qualitatively similar results were obtained with 2-trans-5-trans-octadienoyl-CoA formed by dehydrogenation of 5-trans-octenoyl-CoA. 2-trans-4-trans-Octadienoyl-CoA was a substrate for NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34). A soluble extract of rat liver mitochondria catalyzed the isomerization of 2-trans-5-cis-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA, which upon addition of NADPH, NAD+, and CoA was chain-shortened to hexanoyl-CoA, butyryl-CoA, and acetyl-CoA. Thus we conclude that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the beta-oxidation of polyunsaturated fatty acids.