Flow-induced vasodilation in hamster cremasteric arterioles was investigated with special reference to the roles of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). Arterioles (∼60 µm resting diameter) were cannulated, and suffused with MOPS solution at 37°C (mean intraluminal pressure: 80 cm H<sub>2</sub>O). Step increases in the perfusate flow elicited a dose-dependent vasodilation, which was almost proportional to the increases in calculated wall shear stress (WSS). N<sup>ω</sup>-nitro L-arginine methyl ester ( L-NAME, 100 µ M) reduced the flow-induced vasodilation by ∼50%, whereas indomethacin (10 µ M) produced no significant effect. In the presence of L-NAME, the residual vasodilation was eliminated by treatment with the cytochrome P-450 monooxygenase inhibitor 17-octadecynoic acid (17-ODYA, 50 µ M), sulfaphenazol (10 µ M), tetraethylammonium (TEA, 3 m M; a nonselective Ca<sup>2+</sup>-activated K<sup>+</sup> channel inhibitor), or charybdotoxin (ChTX, 0.1 µ M; intermediate or large conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channel inhibitor). In the absence of L-NAME, the dilation was also reduced by ∼50% by treatment with 17-ODYA, TEA, or ChTX. The residual vasodilation was eliminated by additional treatment with L-NAME. The inhibitor of ATP-sensitive K<sup>+</sup> channels (K<sub>ATP</sub>), glibenclamide, also caused a significant, but partial, reduction of the flow-induced vasodilation. The residual vasodilation was completely reduced by additional treatment with 17-ODYA, but not L-NAME. These findings suggest that in hamster cremaster, higher flow rate produces NO, K<sub>ATP</sub>, and EDHF vasodilation of the arterioles under physiological conditions.