Aggregating human platelets contract isolated rings of canine coronary artery without endothelium, but relax rings with intact endothelium. We performed experiments to identify the substances released from platelets responsible for these effects. The contraction in rings without endothelium was reduced by treating the platelets with thromboxane synthetase inhibitor, dazoxiben, or treating the vessels with the thromboxane-receptor antagonist, SQ 29548. The serotonergic antagonist, methiothepin, also reduced the platelet-induced contraction. The combination of methiothepin plus dazoxiben or SQ 29548 caused a further inhibition. The endothelium-dependent relaxation to platelets during contractions evoked by prostaglandin F2 alpha was nearly abolished by the ADP- and ATP-scavenger, apyrase. It was not inhibited by methiothepin, which antagonizes endothelium-dependent relaxations to serotonin. Thus, both serotonin and thromboxane A2 contribute to the direct activation of coronary smooth muscle by aggregating human platelets, whereas adenine nucleotides are the principal mediators of the endothelium-dependent relaxation.