Shear stress is one of the most important stimulators for the release of endothelium-derived relaxing factors. Although shear stress-induced release of nitric oxide (NO) has been extensively investigated, it remains to be elucidated whether endothelium-derived hyperpolarizing factor (EDHF) contributes to the endothelium-dependent relaxations to shear stress. This study was designed to address this point in the isolated rat mesenteric artery. Large mesenteric arteries (400-500 μm) and resistance mesenteric arteries (150-250 μm) of the rat were precontracted with phenylephrine (at 80 mm Hg of perfusion pressure), and the changes in vessel diameter in response to variable flow (0-300 μl/min) were continuously examined. The relative contributions of vasodilator prostaglandins, NO, and EDHF were analyzed by the inhibitory effects of indomethacin (10− 5 M), N G-nitro-L-arginine (L-NNA, 10− 4 M), and KCl (40 mM), respectively. The shear stress-induced relaxations were totally endothelium dependent in both-sized blood vessels, and the contribution of NO was more prominent in large arteries than in resistance arteries, whereas that of EDHF was noted in both-sized blood vessels. Tetrabutylammonium (a nonselective inhibitor of K channels) almost abolished, whereas the combination of charybdotoxin (an inhibitor of both large-and intermediate-conductance Ca 2+-activated K channels) and apamin (an inhibitor of small-conductance Ca 2+-activated K channels) significantly inhibited the EDHF-mediated component of the shear stress-induced relaxations. These results indicate that EDHF plays an important role in shear stress-induced endothelium-dependent relaxations, where K channels, especially calcium-activated K channels, appear to be involved.