Whereas the actual identity of endothelium-derived hyperpolarizing factor (EDHF) is still not certain, it involves a process requiring the endothelium and eliciting hyperpolarization and relaxation of smooth muscle. It is neither nitric oxide (NO) nor prostacyclin, and its presence has been demonstrated in a variety of vessels. Recent studies in peripheral vessels report that EDHF-mediated dilations were either attenuated or blocked by NO. Studies presented here demonstrate that NO does not block EDHF-mediated dilations in cerebral vessels. Rat middle cerebral arteries were cannulated, pressurized, and luminally perfused. EDHF-mediated dilations were elicited by the luminal application of ATP in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin (inhibitors of NO synthase and cyclooxygenase, respectively). These dilations persisted when S-nitroso-N-acetylpenicillamine, an NO donor, was added exogenously in the presence of L-NAME, or when endogenous NO was present but its cGMP actions were blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylate cyclase. These findings demonstrate that the EDHF response is not suppressed by NO in cerebral vessels and suggests a role for EDHF during normal physiological conditions.