Background: We have recently identified that endothelium-derived hydrogen peroxide (H2O2) is an endothelium- derived hyperpolarlizing factor (EDHF) both in vitro and in vivo and that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role as an EDHF synthase in vitro. In this study, we examined the possible role of Cu/Zn-SOD in the synthesis of EDHF/ H2O2during reactive hyperemia in mouse mesenteric microcirculation in vivo. Methods: Mesenteric arterioles in wild-type and Cu/Zn-SOD-/-mice (n=6 each, 20-60 μm in diameter) were continuously observed by a newly developed pencil-typed intravital microscope during acetylcholine (ACh)-induced vasodilatation and reactive hyperemia (RH, reperfusion after 20 sec of mesenteric artery occlusion) under cyclooxygenase blockade (indomethacin, 5x10-5M, topically administration, TA) under the following 3 conditions; control, NO synthase inhibitor (L-NMMA, 10-4M, TA) and LNMMA+ catalase (PEG-catalase 10,000 U/kg, a specific decomposer of H2O2, intra-arterial administration). Results: In mesenteric arterioles of wild-type mice (n=6), endotheliumdependent relaxations to acetylcholine and vasodilatation during RH were totally resistant to L-NMMA, but were markedly inhibited by L-NMMA+catalase. In mesenteric arterioles of Cu/Zn-SOD-/-mice (n=6), L-NMMA markedly inhibited the relaxations to ACh and vasodilatation during RH. The responses were not inhibited by the addition of catalase. By contrast, endothelium-independent relaxations to sodium nitroprusside (10-4 M, TA), a direct NO donor, were comparable between the two strains in the presence of LNMMA+ catalase. Conclusions: Endothelial Cu/Zn-SOD plays an important role as an EDHF synthase during reactive hyperemia in mouse mesenteric microcirculation in vivo.