Epoxyeicosatrienoic acids (EETs) induce vasorelaxation, probably through G protein-coupled receptors. The identity of these receptors is unclear, but it has been reported that EETs may bind to peroxisome proliferator activated receptors (PPARs) and E-prostanoid (EP) receptors. Therefore, we studied whether PPARs or EP receptors were involved in 14,15-EET-induced vasorelaxation. Isometric tensions of rat mesenteric arteries were measured. The vasorelaxant effect of 14,15-EET was inhibited by NF449 (Gs-protein inhibitor), Rp-cAMP (cAMP antagonist) and KT5720 (PKA inhibitor), suggesting that the effect of 14,15-EET was mediated through Gs protein-coupled receptors which were linked to the cAMP/PKA-dependent pathway. Pretreatments with MK886 (PPARα antagonist) and GW9662 (PPARγ antagonist) did not influence 14,15-EET-induced vasorelaxation. The vasorelaxant effect of 14,15-EET was inhibited by AH6809 (EP2 receptor antagonist), whereas SC19220 (EP1 receptor antagonist), L798106 (EP3 receptor antagonist) and GW627368X (EP4 receptor antagonist) had no effect. The effect of 14,15-EET and the mechanism involved was mimicked by prostaglandin E2 (an EP2 receptor agonist). The 14,15-EET-induced relaxation was slightly potentiated in the presence of indomethacin (cyclooxygenase inhibitor which block PGE2 synthesis). Binding study showed that the amount of 14,15-EET bound to the cell membrane of rat mesenteric arterial smooth muscle cells was much higher than that bound to the nuclear membrane. The binding of 14,15-EET to the cell membrane was attenuated by AH6809 and siRNA against EP2 receptors. In conclusion, our study has demonstrated that 14,15-EET exerts relaxant effects on rat mesenteric arteries, at least partly via the stimulation of EP2 receptors. This subsequently leads to activation of cAMP/PKA-dependent pathway in vascular smooth muscle cells. © 2010 Elsevier Inc.