This study was designed to test whether or not 1) ischemia-reperfusion attenuates endothelium-dependent relaxation of coronary arteries and 2) preconditioning protects the arterial endothelium from ischemia-reperfusion injury. In anesthetized open chest rabbits, branches of the left circumflex artery were exposed to different combinations of the experimental conditions; ischemia (15 minutes), ischemia (15 minutes)-reperfusion (10 minutes), preconditioning ischemia, and pre-conditioning fellowed by ischemia-reperfusion. Preconditioning consisted of 3 occlusions of 2-min duration, each followed by n 5-min reperfusion. Rings of the artery exposed to the experimental condition and of normal left anterior descending coronary artery were prepared and suspended for isometric force measurement in organ chambers containing Krebs Ringer bicarbonate solution. The rings were contracted with 29.6 mM KCI. Ischemia alone did not attenuate endothelium-dependent relaxation by acetylcholine. However, ischemia-reperfusion significantly impaired endothelium-dependent relaxation. Endothelium-independent relaxation by sodium nitroprusside was not impaired by ischemia-reperfusion and the constrictive response to acetylcholine was not altered in reperfused rings without endothelium, compared with control rings. Arterial rings exposed to preconditioning followed by ischemia-reperfusion exhibited impaired endothelium-dependent relaxation by acetyl-choline. However, although preconditioning not fellowed by ischemia-reperfusion, attenuated endothelium-dependent relaxation at low concentrations of acetylcholine, the magnitude of the impairment by preconditioning followed by ischemia-reperfusion was significantly less than that of the impairment by ischemia-reperfusion alone. These data demonstrate that ischemia-reperfusion significantly attenuates endothelium-dependent relaxation by producing endothelial dysfunction and preconditioning Protects the endothelium of coronary arteries from ischemia-reperfusion injury.