Epoxyeicosatrienoic acid (EET) metabolism was studied in endothelial cells to determine whether this tissue may influence their vasoactive properties. Porcine aortic endothelial cells rapidly took up all four EET regioisomers. The uptake of [1-14C]14,15-EET reached a maximum in 15-30 min, and saturation was not observed with concentrations up to 5 microM. More than 70% of the incorporated 14,15-EET was contained in choline and inositol glycerophospholipids, most of it in the form of an EET ester. A metabolite, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), accumulated in the medium during incubation, and products with similar chromatographic properties also were formed from 5,6-, 8,9-, and 11,12-EET. Much of the 14,15-EET taken up was only temporarily retained by the cells, and in 2 h half was released into the medium as 14,15-DHET. Bovine aortic and human umbilical vein endothelial cells also took up 14,15-EET, incorporated it into choline glycerophospholipids, and converted it to 14,15-DHET. These findings suggest that the endothelium may limit the vascular actions of EETs through rapid uptake, hydration, and release of DHETs into the circulation. Some vasoactive effects of EETs may result from their temporary accumulation in endothelial phospholipids involved in stimulus-response coupling.