Activation of the purinergic P2X(7) receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the Delta18 mutant with deleted P2X(7) receptor-specific 18-amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. These results indicate that the P2X(7) receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.