The purpose of the present study was to examine in peripheral arteries whether the endothelium plays a protective role against aggregating platelets and whether the responses to platelets are altered by hypercholesterolemia. Male Yorkshire pigs were fed either a normal diet or a 2% high-cholesterol diet for 10 weeks. Endothelium-dependent responses were examined in vitro. In isolated iliac arteries from control animals, aggregating platelets caused contractions that were significantly inhibited by the endothelium. In rings with endothelium from pigs fed a cholesterol diet, the contractions were augmented and no difference was noted between rings with and rings without endothelium. In rings taken from control pigs contracted with prostaglandin F2 alpha, aggregating platelets caused endothelium-dependent relaxations, which were attenuated by blockade of adenosine diphosphate or serotonin. Adenosine diphosphate and serotonin also caused endothelium-dependent relaxations. In arteries from cholesterol-fed animals, the inhibitory effects of the endothelium to aggregating platelets, adenosine diphosphate, and serotonin were impaired. These experiments indicate that in porcine iliac arteries the endothelium exerts inhibitory effects on the responses to aggregating platelets, which are mediated by adenosine diphosphate and serotonin released from platelets. Hypercholesterolemia impairs the endothelium-dependent relaxations to aggregating platelets caused by reduced relaxations to adenosine diphosphate and serotonin and unmasks the contractions evoked by aggregating platelets. This may increase the level of vascular tone, decrease blood flow and increase platelet aggregation, and eventually facilitate the occurrence of peripheral arterial occlusive disease.