Epoxyeicosatrienoic acids (EETs) are important regulators of vascular tone and homeostasis. Whether they initiate signaling through membrane receptors is unclear. We developed 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), a radiolabeled EET agonist, to characterize EET binding to membranes of U937 cells. 20-I-14,15-EE8ZE stimulated cAMP production in U937 cells with similar potency, but it decreased efficacy compared with 11,12-EET. Maximum cAMP production increased 4.2-fold, with an EC(50) value of 9 muM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC(50) value. Both 20-I-14,15-EE8ZE agonist activities were blocked by the EET antagonist 14,15-epoxyeicosa-5(Z)enoic acid (14,15-EE5ZE). Specific 20-(125)I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4 degrees C. The binding was saturable, reversible, and exhibited K(D) and B(max) values of 11.8 +/- 1.1 nM and 5.8 +/- 0.2 pmol/mg protein, respectively. Pretreatment of the membranes with guanosine 5'-O-(3-thio)triphosphate reduced the B(max) in a concentration-related manner. 20-(125)I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE approximately 14,15-EET >> 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-(125)I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.