Changes in isometric tension due to intra- or extraluminal addition of vasoactive agents were determined in isolated perfused segments of canine left circumflex coronary artery. Segments denuded of endothelium were more sensitive to the contractile action of 5-hydroxytryptamine and potassium during intraluminal addition. In segments with intact endothelium, the sensitivity to intraluminal, but not extraluminal, 5-hydroxytryptamine was decreased in comparison to denuded segments; that to potassium was unchanged. In segments with intact endothelium contracted with prostaglandin F2 alpha, intraluminal, but not extraluminal, acetylcholine, adenosine diphosphate, or thrombin caused relaxation. Intraluminal 5-hydroxytryptamine and aggregating platelets caused relaxation or attenuated contractions in a majority of vessels studied; extraluminal addition caused only contractions. Thus the endothelium is responsible for opposite smooth muscle responses to intra- versus extraluminal vasoactive substances released from aggregating platelets. During intraluminal thrombosis the endothelium may inhibit smooth muscle contraction by responding to 5-hydroxytryptamine and adenosine diphosphate released from platelets and to thrombin; where the endothelium is damaged, the luminal aspect of the blood vessel wall, which is more sensitive to 5-hydroxytryptamine, may become the site of coronary spasm.