Pannexins (Panx) form ATP release channels and it has been proposed that they play an important role in the regulation of vascular tone. However, distribution of Panx across the arterial vasculature is not documented. We tested antibodies against Panx1, Panx2 and Panx3 on human embryonic kidney cells (which do not endogenously express Panx proteins) transfected with plasmids encoding each Panx isoform and Panx1–/–mice. Each of the Panx antibodies was found to be specific and was tested on isolated arteries using immunocytochemistry. We demonstrated that Panx1 is the primary isoform detected in the arterial network. In large arteries, Panx1 is primarily in endothelial cells, whereas in small arteries and arterioles it localizes primarily to the smooth muscle cells. Panx1 was the predominant isoform expressed in coronary arteries, except in arteries less than 100 µm where Panx3 became detectable. Only Panx3 was expressed in the juxtaglomerular apparatus and cortical arterioles. The pulmonary artery and alveoli had expression of all 3 Panx isoforms. No Panx isoforms were detected at the myoendothelial junctions. We conclude that the specific localized expression of Panx channels throughout the vasculature points towards an important role for these channels in regulating the release of ATP throughout the arterial network.