Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-α (PPARα). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARα activator in a COS-7 cell expression system. Incubation with 10 μM 14,15-DHET produced a 12-fold increase in PPARα-mediated luciferase activity, an increase similar to that produced by the PPARα agonist Wy-14643 (20 μM). Although 10 μM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5(Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPARα. However, PPARα was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[³H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[³H]EET. Binding studies indicated that 14,15-[³H]DHET binds to the ligand binding domain of PPARα with a K_d of 1.4 μM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARα-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPARα.