Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X₇ receptor (P2X₇R) 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 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X₇R antagonist, prevented current activation by 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (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 Src homology 3 death domain of the COOH-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 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 P2X₇R.