ATP-gated P2X channels are the simplest of the three families of transmitter-gated ion channels. Some P2X channels display a time- and activation-dependent change in permeability as they undergo the transition from the relatively Na +-selective I 1 state to the I 2 state, which is also permeable to organic cations. We report that the previously reported permeability change of rat P2X 2 (rP2X 2) channels does not occur at mouse P2X 2 (mP2X 2) channels expressed in oocytes. Domain swaps, species chimeras, and point mutations were employed to determine that two specific amino acid residues in the cytosolic tail domain govern this difference in behavior between the two orthologous channels. The change in pore diameter was characterized using reversal potential measurements and excluded field theory for several organic ions; both rP2X 2 and mP2X 2 channels have a pore diameter of ∼11 Å in the I 1 state, but the transition to the I 2 state increases the rP2X 2 diameter by at least 3 Å. The I 1 to I 2 transition occurs with a rate constant of ∼0.5 s −1. The data focus attention on specific residues of P2X 2 channel cytoplasmic domains as determinants of permeation in a state-specific manner.