Objective-Coronary flow is closely correlated to the myocardial metabolic demand. We tested the hypothesis that hydrogen peroxide (H 2 O 2 ) derived from beating hearts mediates metabolic coronary microvascular dilation. Methods and Results-We used a bioassay method in which an isolated microvessel is placed on a beating heart to detect myocardium-derived vasoactive mediators. A rabbit coronary arterial microvessel (detector vessel [DV], nϭ25) was pressurized and placed on a canine beating heart. After intrinsic tone of DV had developed, we observed DV at rest (heart rate, 120 bpm) and during tachypacing (heart rate, 240 bpm) using an intravital microscope equipped with a floating objective. The tachypacing produced DV dilation by 8.2% (PϽ0.01 versus baseline), and the dilation was abolished by cell-impermeable catalase (a H 2 O 2 scavenger, 500 U/mL). We performed myocardial biopsy at rest and tachypacing. The biopsy specimens were loaded with 2Ј,7Ј-dichlorodihydrofluorescein diacetate (10 mol/L) to visualize H 2 O 2, and observed with confocal microscopy. Dichlorofluorescein fluorescence was diffusely identified in the myocardium and the tachypacing increased the fluorescence intensity (PϽ0.01). Exogenous H 2 O 2 caused vasodilation of arterial microvessels in vitro in a concentration-dependent manner that was abolished by catalase. Conclusions-H 2 O 2 derived from the beating heart mediates tachypacing-induced metabolic coronary vasodilation in vivo.(Arterioscler Thromb Vasc Biol. 2007;27:1057-1063.)Key Words: coronary circulation Ⅲ myocardium Ⅲ reactive oxygen species Ⅲ tachycardia Ⅲ vasodilation C oronary blood flow is linearly correlated to the myocardial oxygen consumption. 1,2 The tight coupling between the cardiac metabolism and the flow conductance underlies the widely accepted assumption that the myocardium-derived vasoactive factor, so-called metabolic factor, rapidly regulates the vascular tone of coronary microvessels for matching the coronary flow to the cardiac metabolic state. However, the metabolic factor that is transmitted from the myocardium to the coronary microvessels has not been identified yet. Although there are many candidates for the mediators such as adenosine, prostanoids, autacoids, nitric oxide (NO), factors activating potassium channels, and so on, no substance solely explains the metabolic microvascular dilation. 3,4 Although excessive reactive oxygen species (ROS) are produced in various pathological conditions such as ischemia/reperfusion and cardiac failure 5,6 and are often hazardous for living organisms, increasing evidence has shown that ROS also play important roles as biological signals that mediate physiological phenomena at low concentrations. 7,8 In the field of vascular biology, hydrogen peroxide (H 2 O 2 ) has been known as one of the possible endothelium-derived hyperpolarizing factors. 9 The cardiac myocyte is another major source of ROS. 5,10 Superoxide (O 2 Ϫ ⅐ ) and H 2 O 2 are produced as byproducts of electron transfer reactions during normal aerobic meta...