ATP is an important endogenous mediator in the cardiovascular system. It induces endothelium dependent vasodilation, but the precise receptor pathway activated in this response is currently under debate. We have used traditional bioassay techniques to show that ATP-induced vasodilation in mesenteric vessels is endothelium-dependent. Furthermore, ATP-induced vasodilation was inhibited by both suramin and 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), consistent with a P2X1-, P2X2-, or P2X3-mediated event and was not potentiated by ivermectin, indicating that these responses were not P2X4 receptor-mediated. ATP did not induce vasodilation in vessels from P2X –/–1 mice, confirming an absolute requirement for this receptor. Finally, in pure cell populations of mouse mesenteric artery endothelial cells, we show that P2X1 mRNA is specifically expressed. However, in line with observations in the brain, the P2X1 present in endothelial cells does not seem to be recognized by conventional antibodies. Together, these results show that ATP-induced vasodilation is mediated by P2X1 receptor activation on mesenteric arterial endothelial cells. These observations establish a critical role for P2X1 receptors in the ATP vasodilator pathway.