The myogenic tone is intrinsic to vascular tissue and plays an important role in determining basal coronary resistance. However, the effect of the beating heart on the myogenic tone is unknown. We investigated the effects of myocardium-derived vasoactive factors on the myogenic tone of the coronary microvessels in the resting condition and during increased metabolism. Pressurized isolated coronary vessels (detector vessel: DV) of rabbits (n=33, maximal inner diameter 201±8 µm) were gently placed on the beating hearts of anesthetized dogs and observed with an intravital microscope equipped with a floating objective. To shut off the myocardium-derived vasoactive signals, a plastic film was placed between DV and the heart. The intravascular pressure was changed from 120 to 60 cm H₂O and pressure-diameter curves were obtained with and without the contact of DV and the myocardium. The direct contact shifted the pressure-diameter curve upward (p<0.05 vs without contact) and the myogenic tone was reduced by 40%. When endothelium of DV was denuded, the shift persists, but the degree of the shift was reduced to 10% (p<0.05 vs with endothelium). The shift was abolished by glibenclamide, an ATP-sensitive potassium (K⁺_ATP) channel blocker. A similar upward shift was induced by rapid pacing, but the shift was not blocked by glibenclamide. We conclude that the beating myocardium counteracts the myogenic tone by releasing transferable vasoactive signals that effect on the endothelium and the vascular smooth muscle, and that the signals are solely mediated by the activation of K⁺_ATP channels, unlike the rapid pacing-induced vasoactive factors.