Erythrocytes serve as reservoirs for cis- and trans-epoxyeicosatrienoic acids (EETs). Incubation of rat red blood cells (RBCs) with cis- and trans-EETs produces threo- and erythro-dihydroxyeicosatrienoic acids, respectively. The V(max) of EET hydrolysis by rat intact RBCs (2.35 +/- 0.24 pmol/min/10(8) RBCs for 14,15-trans-EET) decreased by approximately 2 to 3-fold sequentially from 14,15-, 11,12- to 8,9-EETs for both cis- and trans-isomers. The V(max) of trans-EET hydrolysis by RBCs is approximately 2 to 3 times that of the corresponding cis-EETs. Incubation of EETs with recombinant murine soluble epoxide hydrolase (sEH) yielded the same geometric and regio preferences of EET hydrolysis as with rat intact RBCs. The principal epoxide hydrolase activity for EET hydrolysis (approximately 90%) is present in the erythrocyte cytosol. Western blots of sEH suggested a concentration of sEH protein to be approximately 2 microg/mg protein or 0.4 microg/10(9) RBCs. The apparent K(m) values of EETs were between 1 and 2 microM, close to the K(m) for purified sEH as reported. Erythrocyte hydration of cis- and trans-EETs was blocked by sEH inhibitors, 1,3-dicyclohexylurea and 4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid. Erythrocyte sEH activity was inhibited more than 80% by 0.2% bovine serum albumin in the buffer. Preferred hydrolysis of 14,15-EETs and trans-epoxides characterizes sEH activity in RBCs that regulates the hydrolysis and release of cis- and trans-EETs in the circulation. Inhibition of sEH has produced antihypertensive and antiinflammatory effects. Because plasma trans-EETs would increase more than cis-EETs with sEH inhibition, the potential roles of trans-EETs and erythrocyte sEH in terms of circulatory regulation deserve attention.