In this study, we have investigated the mechanism of ADP-induced relaxation of porcine coronary artery (PCA) rings. The P2Y receptor agonists ADP and ADPS produced concentration-dependent relaxation of endothelium-denuded PCA smooth muscle with pD₂ values of 5.3 and 4.9, respectively. RT-polymerase chain reaction (RT-PCR) and immunoblotting demonstrated mRNA and protein expression of P2Y₁ and A_2A adenosine receptors in the PCA. The nonselective P2 antagonist PPADS or the P2Y₁-selective antagonist MRS2179 failed to alter ADP- or ADPS-induced relaxations. Relaxations to ADP were, however, blocked by the A_2A adenosine receptor-selective antagonists ZM241385 and SCH58261 (apparent pK_B values of 9.2 and 8.9, respectively). We excluded roles for direct occupancy of A_2A adenosine receptors by ADP or ADPS as well as metabolism to adenosine as mechanisms for ADP-evoked relaxations. However, ADP responses were significantly enhanced in the presence of the ENT1 nucleoside transporter inhibitors dipyridamole and NBTI and were significantly inhibited by adenosine deaminase, indicating a role for extracellular adenosine. Suprafusion of [³H]-adenine-labeled PCA segments showed that ADP induced the release of a number of purines, including adenosine. These data suggest that ADP mediates relaxation of the PCA via a novel mechanism that involves adenine nucleotide-evoked adenosine release and the subsequent activation of A_2A receptors.--Rayment, S. J., Ralevic, V., Barrett, D. A., Cordell, R. and Alexander, S. P. H. A novel mechanism of vasoregulation: ADP-induced relaxation of the porcine-isolated coronary artery is mediated via adenosine release