The mechanisms underlying endothelium-dependent hyperpolarizing factor (EDHF) in the middle cerebral artery (MCA) remain largely unresolved. In particular, very little is known regarding the way in which the signal is transmitted from the endothelium to the smooth muscle. The present study tested the hypothesis that direct communication via myoendothelial gap junctions contributes to the EDHF response in the male rat MCA. EDHF-mediated dilations were elicited in rat MCAs by the luminal application of ATP or UTP in the presence of L-NAME and indomethacin. Maximum dilation to luminal ATP (10^-4M) was reduced significantly following incubation with a gap peptide cocktail (9±4%; n=6) compared with a scrambled gap peptide cocktail (99±1%; n=6; p<0.05). A gap peptide cocktail was without effect on the amplitude of endothelial cell hyperpolarization in response to UTP (3x10^-5M; 22±3mV versus 22±1mV; n=4) while smooth muscle cell hyperpolarization was significantly attenuated (17±1mV versus 6±1mV; n=4; p=0.004). Connexin 37 (Cx37) was localized to the smooth muscle and Cx43 was localized to the endothelium, while Cx40 was found in both endothelium and smooth muscle. Electron microscopy revealed the existence of frequent myoendothelial junctions (MEJs). The total number of MEJs/µm MCA sectioned was 2.5±0.5. Our results suggest that myoendothelial communication contributes to smooth muscle cell hyperpolarization and EDHF dilation in male rat MCA.