Cytochrome P-450-derived epoxyeicosatrienoic acids (EETs) are avidly incorporated into and released from endothelial phospholipids, a process that results in potentiation of endothelium-dependent relaxation. EETs are also rapidly converted by epoxide hydrolases to dihydroxyeicosatrienoic acid (DHETs), which are incorporated into phospholipids to a lesser extent than EETs. We hypothesized that epoxide hydrolases functionally regulate EET incorporation into endothelial phospholipids. Porcine coronary artery endothelial cells were treated with an epoxide hydrolase inhibitor, 4-phenylchalcone oxide (4-PCO, 20 micromol/l), before being incubated with (3)H-labeled 14,15-EET (14,15-[(3)H]EET). 4-PCO blocked conversion of 14,15-[(3)H]EET to 14,15-[(3)H]DHET and doubled the amount of radiolabeled products incorporated into cell lipids, with >80% contained in phospholipids. Moreover, pretreatment with 4-PCO before incubation with 14,15-[(3)H]EET enhanced A-23187-induced release of radiolabeled products into the medium. In contrast, 4-PCO did not alter uptake, distribution, or release of [(3)H]arachidonic acid. In porcine coronary arteries, 4-PCO augmented 14,15-EET-induced potentiation of endothelium-dependent relaxation to bradykinin. These data suggest that epoxide hydrolases may play a role in regulating EET incorporation into phospholipids, thereby modulating endothelial function in the coronary vasculature.