5-Hydroxytryptamine caused contractions of isolated canine coronary artery rings. These contractions were larger in the absence of the endothelium, whereas those caused by phenylephrine, potassium chloride, and prostaglandin F2 alpha were not. When coronary arteries were contracted with prostaglandin F2 alpha, 5-hydroxytryptamine caused relaxation in some rings with endothelium but only further contraction in all rings without endothelium. The inhibitory action of 5-hydroxytryptamine mediated by the endothelium was unaffected by blockade of monoamine oxidase or cyclooxygenase. In rings with endothelium, aggregating platelets, which released 5-hydroxytryptamine and thromboxane A2, caused relaxation. The relaxations caused by 5-hydroxytryptamine and aggregating platelets were antagonized by methysergide but not by ketanserin. These observations suggest that the response to 5-hydroxytryptamine is the net result of a direct contractile action on coronary smooth muscle and an inhibitory action mediated by the endothelium. In some vessels the endothelium-dependent inhibitory responses to aggregating platelets may be mediated in part by released 5-hydroxytryptamine. The serotonergic receptors on endothelial cells may be of a different subtype than those mediating contractions of the smooth muscle cells.