Moderate consumption of wine is associated with cardiovascular protection most likely by increasing the endothelial formation of nitric oxide (NO). The present study investigated whether red wine polyphenolic compounds (RWPCs) increase the formation of endothelium-derived hyperpolarizing factor (EDHF) in arteries and, if so, to characterize the underlying mechanism. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension and membrane potential in the presence of indomethacin and N(omega)-nitro-L-arginine. RWPCs caused pronounced endothelium-dependent relaxations and hyperpolarizations, which were reduced by the combination of charybdotoxin plus apamin (two inhibitors of EDHF-mediated responses). Both responses to RWPCs were also reduced by antioxidants, membrane permeant analogues of superoxide dismutase, and diphenylene iodonium, an inhibitor of flavin-dependent enzymes. RWPCs induced the formation of superoxide in cultured endothelial cells. These findings demonstrate that RWPCs cause EDHF-mediated relaxations of coronary arteries, which are critically dependent on a redox-sensitive mechanism involving a flavin-dependent enzyme.