Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by two carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06±0.01 and 6.18±0.60 µg/mg protein and adrenic acid concentrations of 0.29±0.01 and 1.56±0.16 µg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic, U46619, adrenic acid (10^-9-10^-5M) induced concentration-related relaxations (maximal relaxation=83±4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the potassium channel inhibitor, iberiotoxin (100nM) and inhibited by the cyclooxygenase inhibitor, indomethacin (10µM, maximal relaxation=53±4%) and the cytochrome P450 inhibitor, miconazole (10µM, maximal relaxation=52±5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxyeicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET (16,17-, 13,14-, 10,11- and 7,8- (10^-9-10^-5M)) induced similar concentration-related relaxations (maximal relaxations averaged 83±3%). Adrenic acid (10^-6M) and DH-16,17-EET (10^-6M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10^-8-10^-6M) activated iberiotoxin-sensitive, whole-cell potassium currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome 450 metabolites. The adrenic acid metabolite, 16,17-DH-EET activates smooth muscle potassium channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium-derived hyperpolarizing factors in the coronary circulation.