T he purpose of this study was to determine the effect of vasoactive substances released by aggregating platelets on adrenergic neurotransmission in canine coronary arteries. Isometric tension was recorded in isolated ring segments of coronary artery denuded of endothelium and the release of [3H]norepinephrine was measured from strips of coronary artery preincubated with the radiolabeled transmitter. Transmural electrical field stimulation and exogenously added norepinephrine caused beta adrenergic relaxations of coronary rings contracted by prostaglandin F2 alpha. In coronary rings further contracted by the addition of aggregating platelets in numbers less than that present in blood, the response to electrical stimulation was inhibited and the sensitivity to norepinephrine reduced. Micromolar concentrations of adenosine diphosphate, adenosine triphosphate, and 5-hydroxytryptamine were released by platelets under these experimental conditions. The reduced response to electrical stimulation was in part due to inhibition of the stimulated release of [3H]-norepinephrine. The combination of the serotonergic antagonist, methiothepin, and the purinergic antagonist, theophylline, attenuated the inhibition of the responses of coronary rings; either antagonist alone failed to do so, but did significantly block the reductions caused by 5-hydroxytryptamine and adenosine diphosphate, respectively. In addition, only the combination of the two antagonists significantly attenuated the inhibition of norepinephrine release caused by platelets. These data suggest that both adenine nucleotides and 5-hydroxytryptamine are important mediators of the prejunctional and postjunctional inhibition of coronary beta adrenergic neurotransmission caused by platelets.