A remarkable feature of a sustained P2X7 receptor activation by ATP is the induction of a large cytolytic pore permeable to cations up to 700 Da. Whether the cytolytic pore is formed by the homotrimeric P2X7 receptor itself or by the downstream activation of secondary channel proteins such as pannexin-1 is not finally settled. Here, we assessed the specificity of the functional and physical interaction between pannexin-1 and the P2X7 receptor. We found that recombinant pannexin-1 and the P2X7 receptor expressed in Xenopus laevis oocytes form disulfide-bonded oligomers in the endoplasmic reticulum (ER). However, at the level of the plasma membrane, we could not detect disulfide-bonded oligomers between pannexin-1 and hP2X7 nor any non-covalently linked physically stable complexes between the two proteins. This suggests that the disulfide-bonded oligomers are retained in the ER by the ER quality control system. Also by using two-electrode voltage-clamp electrophysiology, we obtained no evidence for a functional interaction of human or murine P2X7 receptors and pannexin-1 before and during P2X7 receptor activation by ATP. Although our data confirm that certain forms of P2X7 receptors and pannexin-1 interact physically stably with each other, they argue against the view that this interaction takes place at the cell membrane and that pannexin-1 represents the pore-forming principle activated through P2X7 receptors.We thank the German Research foundation (Deutsche Forschungsgemeinschaft, DFG) for financial support (grants Ma1581/15-1 and Schm536/9-1).