Pannexin1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X₇ receptor activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X₇R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X₇R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X₇R stimulation or by membrane depolarization was blocked by Panx1 siRNA as well as with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X₇R antagonist, prevented current activation by BzATP. Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X7) peptides, provided evidence that the SH3-death domain of the C-terminus of the P2X₇R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 µM TAT-P2X7 peptide caused 50% reduction in Src binding to the P2X₇R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X7 peptide and by the Src tyrosine inhibitor PP2, and that these compounds prevented both large conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X₇R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X₇ receptor.