Unsaturated fatty acids with odd-numbered double bonds, e.g. oleic acid, can be degraded by beta-oxidation via the isomerase-dependent pathway or the reductase-dependent pathway that differ with respect to the metabolism of the double bond. In an attempt to elucidate the metabolic functions of the two pathways and to determine their contributions to the beta-oxidation of unsaturated fatty acids, the degradation of 2-trans,5-cis-tetradecadienoyl-CoA, a metabolite of oleic acid, was studied with rat heart mitochondria. Kinetic measurements of metabolite and cofactor formation demonstrated that more than 80% of oleate beta-oxidation occurs via the classical isomerase-dependent pathway whereas the more recently discovered reductase-dependent pathway is the minor pathway. However, the reductase-dependent pathway is indispensable for the degradation of 3,5-cis-tetradecadienoyl-CoA, which is formed from 2-trans,5-cis-tetradecadienoyl-CoA by delta(3),delta(2)-enoyl-CoA isomerase, the auxiliary enzyme that is essential for the operation of the major pathway of oleate beta-oxidation. The degradation of 3,5-cis-tetradecadienoyl-CoA is limited by the capacity of 2,4-dienoyl-CoA reductase to reduce 2-trans,4-trans-tetradecadienoyl-CoA, which is rapidly formed from its 3,5 isomer by delta(3,5),delta(2,4)-dienoyl-CoA isomerase. It is concluded that both pathways are essential for the degradation of unsaturated fatty acids with odd-numbered double bonds inasmuch as the isomerase-dependent pathway facilitates the major flux through beta-oxidation and the reductase-dependent pathway prevents the accumulation of an otherwise undegradable metabolite.