In this study, we have investigated the mechanism of ADP‐induced relaxation of porcine coronary artery (PCA) rings. The P2Y receptor agonists ADP and ADPβS 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 ADPβS‐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 ADPβS 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 FASEB J. 21, 577–585 (2007)