Endothelial cells release several compounds, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF), that mediate the vascular effects of vasoactive hormones. The identity of EDHF remains unknown. Since arachidonic acid causes endothelium-dependent relaxations of coronary arteries through its metabolism to epoxyeicosatrienoic acids (EETs) by cytochrome P₄₅₀, we wondered if the EETs represent EDHFs. Precontracted bovine coronary arteries relaxed in an endothelium-dependent manner to methacholine. The cytochrome P₄₅₀ inhibitors, SKF 525A and miconazole, significantly attenuated these relaxations. They were also inhibited by tetraethylammonium (TEA), an inhibitor of Ca²⁺-activated K⁺ channels, and by high [K⁺]_o (20 mmol/L). Methacholine also caused hyperpolarization of coronary smooth muscle (−27±3.9 versus −40±5.1 mV), which was completely blocked by SKF 525A and miconazole. In vessels prelabeled with [³H]arachidonic acid, methacholine stimulated the release of 6-ketoprostaglandin F_1α, 12-HETE, and the EETs. Arachidonic acid relaxed precontracted coronary arteries, which were also blocked by TEA, charybdotoxin, another Ca²⁺-activated K⁺ channel inhibitor, and high [K⁺]_o. 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET relaxed precontracted coronary vessels (EC₅₀, 1×10⁻⁶ mol/L). The four regioisomers were equally active. TEA, charybdotoxin, and high [K⁺]_o attenuated the EET relaxations. 11,12-EET hyperpolarized coronary smooth muscle cells from −37±0.2 to −59±0.3 mV. In the cell-attached mode of patch clamp, both 14,15-EET and 11,12-EET increased the open-state probability of a Ca²⁺-activated K⁺ channel in coronary smooth muscle cells. This effect was blocked by TEA and charybdotoxin. These data support the hypothesis that the EETs are EDHFs.