The degradation of 2-trans,5-cis-tetradecadienoylCoA, a metabolite of oleic acid, by the purified complex of fatty acid oxidation from Escherichia coli was studied to determine how much of the metabolite is converted to 3,5-cis-tetradecadienoyl-CoA and thereby diverted from the classical, isomerase-dependent pathway of oleate -oxidation. Approximately 10% of the 2,5-intermediate was converted to the 3,5-isomer. When the latter compound was allowed to accumulate, it strongly inhibited the flux through the main pathway. Since ⌬ 3,5 ,⌬ 2,4 -dienoyl-CoA isomerase was not detected in E. coli cells grown on oleate, the 3,5-intermediate cannot be metabolized via the reductase-dependent pathway. However, it was hydrolyzed by a thioesterase, which was most active with 3,5-cis-tetradecadienoyl-CoA as substrate and which was induced by growth of E. coli on oleate. An analysis of fatty acids present in the medium after growth of E. coli on oleate revealed the presence of 3,5-tetradecadienoate, which was not detected after cells were grown on palmitate or glucose. Altogether, these data prompt the conclusion that oleate is mostly degraded via the classical, isomerase-dependent pathway in E. coli but that a small amount of 2-trans,5-cistetradecadienoyl-CoA is diverted from the pathway via conversion to 3,5-cis-tetradecadienoyl-CoA by ⌬ 3 ,⌬ 2 -enoyl-CoA isomerase. The 3,5-intermediate, which would strongly inhibit -oxidation if allowed to accumulate, is hydrolyzed, and the resultant 3,5-tetradecadienoate is excreted into the growth medium. This study provides evidence for the novel function of a thioesterase in -oxidation.