Recent studies on the P2X₇ receptor in 2BH4 cells and peritoneal macrophages have demonstrated that raising intracellular Ca²⁺ concentration induces a pore opening similar to P2X₇ receptor pore. Herein, we have investigated whether the pore activated by raises in intracellular Ca²⁺ concentration is associated to P2X7 receptor. Using patch clamp in cell attached, whole cell configuration and dye uptake, we measured the pore opening in cell types that express the P2X₇ receptor (2BH4 cells and peritoneal macrophages), and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10µM) increased intracellular free Ca²⁺ concentration and induced pore formation with conductance of 421 ± 14 pS, t_1/2 for ethidium bromide (EB) uptake of 118 ± 17 s, and t_1/2 for Lucifer yellow (LY) of 122 ± 11 s. P2X₇ receptor antagonists did not block this effect. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore induced effect following increase in intracellular Ca²⁺ concentration. However, 5-(N,N-hexamethylene)- amiloride (HMA), a P2X₇ receptor pore blocker, inhibited the induced pore. Moreover, intracellular signalling modulators, such as calmodulin, phospholipase-C (PLC), mitogen-activated protein kinase (MAPK), and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by rise of intracellular Ca²⁺, at the doses used by using lactate dehydrogenase (LDH) release assay. In conclusion, increased mobilization of intracellular Ca⁺² induces a novel membrane pore pharmacologically different from the P2X₇ associated pore and hemichannel pore.