The objectives of this study were to test the hypotheses that 1) endothelium-dependent regulation of coronary arteriolar reactivity is impaired after ischemia and reperfusion, and 2) preconditioning protects the arteriolar endothelium from reperfusion injury. In anesthetized open-chest dogs, coronary arteriolar diameters (30-110 microns) were measured in the beating heart using intravital microscopy during fluorescent stroboscopic epi-illumination in three groups: 1) control, 2) ischemia and reperfusion: 60-min occlusion and 120-min reperfusion of the left circumflex coronary artery, and 3) preconditioning: 10-min occlusion and reperfusion preceding ischemia-reperfusion. To evaluate endothelial reactivity, the diameter responses of coronary arterioles to the endothelium-dependent vasodilators, acetylcholine and serotonin, were assessed. Ischemia and reperfusion significantly reduced the increase in diameter by serotonin (0 +/- 2 vs. 11 +/- 3% change in controls; P < 0.05) and acetylcholine (7 +/- 2 vs. 20 +/- 2% in controls; P < 0.05). In contrast, preconditioning preserved the dilation to both serotonin (6 +/- 1%) and acetylcholine (24 +/- 3%; both NS vs. control; P < 0.05 vs. ischemia and reperfusion). Dilation by the endothelium-independent vasodilator, papaverine, was similar in the three groups, indicating similar levels of vasodilatory reserve and suggesting that the impaired dilation to acetylcholine and serotonin after ischemia and reperfusion was not due to nonspecific damage to vascular smooth muscle. These data demonstrate that ischemia-reperfusion significantly attenuates endothelium-dependent vasodilation of coronary arterioles in the intact beating heart. Furthermore, preconditioning reduces the endothelial dysfunction of coronary arterioles after ischemia-reperfusion.