P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca 2+ permeabilities that are amongst the highest of all ligand-gated channels ( Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca 2+ permeability of the P2X 2 receptor ( Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca 2+ currents ( Pf%) of the rat and human P2X 1 and P2X 4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X 1 receptor but had no effect on the lower Pf% of the P2X 2 receptor, suggesting that ionized side chains regulate the Ca 2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X 1 and P2X 4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X 2 receptor. Taken together, the data suggest that COO − side chains provide an electrostatic force that interacts with Ca 2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X 1 and P2X 4 receptors is conserved in the P2X 3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His 45 increased Pf% of the P2X 3 channel, suggesting that this positive charge masks the facilitation of Ca 2+ flux by the neighboring Glu 46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca 2+ permeability and flux of some P2X receptors.