We have recently demonstrated that endogenous hydrogen peroxide (H₂O₂) plays an important role in coronary autoregulation in vivo. However, the role of H₂O₂ during coronary ischemia-reperfusion (I/R) injury remains to be examined. In this study, we examined whether endogenous H₂O₂ also plays a protective role in coronary I/R injury in dogs in vivo. Canine subepicardial small coronary arteries (<100 µm) and arterioles (<100 µm) were continuously observed by microscope during coronary I/R (90/60 min) under cyclooxygenase blockade (n=50). Coronary vascular responses to endothelium-dependent vasodilators (acetylcholine) were examined before and after I/R under the following 7 conditions; control, nitric oxide synthase (NOS) inhibitor (L-NMMA), catalase (a decomposer of H₂O₂), 8-sulfophenyltheophylline (8-SPT, an adenosine receptor blocker), L-NMMA+catalase, L-NMMA+tetraethylammonium (TEA, an inhibitor of large conductance K_Ca channels), and L-NMMA+catalase+8-SPT. Coronary I/R significantly impaired the coronary vasodilatation to acetylcholine in both-sized arteries (both P<0.01) and L-NMMA reduced the small arterial vasodilatation (both P<0.01), whereas it increased (P<0.05) the acetylcholine-induced coronary arteriolar vasodilatation with fluorescent H₂O₂ production after I/R. Catalase increased the small arterial vasodilatation (P<0.01) with fluorescent NO production and increased eNOS expression, whereas it decreased the arteriolar response after I/R (P<0.01). L-NMMA+catalase, L-NMMA+TEA, or L-NMMA+catalase+8-SPT further decreased the coronary vasodilatation in both-sized arteries (both P<0.01). L-NMMA+catalase (51±1%), L-NMMA+TEA (50±1%) and L-NMMA+catalase+8-SPT (60±1%) significantly increased myocardial infarct area compared with other 4 groups (control, 42±1%; L-NMMA, 44±1%; catalase, 43±2%; 8-SPT, 41±1% all P<0.01). These results indicate that endogenous H₂O₂, in cooperation with NO, plays an important cardioprotective role in coronary I/R injury in vivo.