ATP co-released with norepinephrine (NE) from cardiac sympathetic nerve terminals increases NE exocytosis via a positive feedback mechanism. Since excessive NE release is an established cause of dysfunction in ischemic heart disease, we studied the role of endogenous and exogenous nucleotidases as part of the development of a novel therapeutic approach to myocardial ischemia and its consequences. Using sympathetic nerve terminals isolated from guinea pig heart (cardiac synaptosomes), we previously established that activation of ATP-gated ionotropic presynaptic P2X purinoceptors (P2XR) promotes exocytosis of NE. This effect was increased by inhibition of the endogenous ecto-nucleotidase (E-NTPDase1/CD39) and decreased by solCD39, a recombinant form of human E-NTPDasel/CD39. Furthermore, depolarization of synaptosomes with K+ also evoked NE exocytosis. This was potentiated by inhibition of E-NTPDase1 with ARL67156 (a selective E-NTPDase inhibitor), and attenuated by addition of solCD39. These findings indicated that ATP released by depolarization of sympathetic terminals enhances NE exocytosis, and that E-NTPDase1 plays an important modulatory role in adrenergic neurotransmission in the heart. In the present study, we investigated the role of E-NTPDasel in exocytosis of NE and ATP in porcine as well as human heart. Cardiac E-NTPDase1 is selectively localized in human and porcine cardiac neurons, and depolarization of cardiac tissue elicits an ω-conotoxin-inhibitable release of both NE and ATP. Inhibition of E-NTPDase1 with ARL67156 markedly potentiated release of ATP, demonstrating that E-NTPDase1 is a major control system for ATP availability at sympathetic nerve endings. Importantly, when ATP exocytosis was increased via inhibition of E-NTPDase1, NE exocytosis increased in parallel. Moreover, when ATP release was reduced by administration of solCD39, NE exocytosis was markedly diminished. Thus, ATP regulates NE exocytosis by activating presynaptic P2XR. This concept is further validated by our finding that the strong correlation between ATP and NE release was abolished by the P2XR antagonist PPADS. We conclude that released ATP governs NE exocytosis by activating presynaptic P2XR and that this is controlled by E-NTPDase1/CD39. Clinically, excessive NE release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, solCD39 may offer a novel therapeutic approach to ischemic complications in the myocardium in addition to its role as an anti-thrombotic agent.