Stimulation of P2X₇ receptor by ATP induces cell membrane depolarization, increase in intracellular Ca²⁺ concentration and, in most cases, permeabilization of cell membrane to molecules up to 900 Daltons. After the activation of P2X₇ at least two phenomena occur: the opening of low conductance (8 pS) cationic channels, and the pore formation. Currently, at least two conflicting hypotheses have been postulated to reconcile these findings: 1) the P2X₇ pore is formed as a result of gradual permeability increase (dilation) of cationic channels; 2) the P2X₇ pore represents a distinct "channel", possibly activated by a second messenger, not directly by extracellular nucleotides. In this study, we investigated whether or not second messengers would be necessary to open the pore associated with P2X₇ receptor in cells that express the pore activity by using the patch clamp technique in whole cell and cell attached configurations in conjunction with fluorescent imaging. In peritoneal macrophages and 2BH4 cells, we detected permeabilization and single channel currents by cell attached configuration when ATP was applied outside the membrane patch, in a condition which oxidized ATP and Lucifer Yellow was maintained within the pipette. Our data support Ca²⁺ as a second messenger associated with pore formation since the permeabilization depended on the presence of intracellular Ca²⁺ and was blocked by BAPTA-AM. In addition, MAPK inhibitors (SB 203580 and PD 98059) blocked the permeabilization and single channel currents in these cells. Together our data indicate that the P2X₇ pore depends on second messengers, such as Ca²⁺ and MAP kinases.