Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426-450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K(+) versus Na(+). ASIC1a shows a selectivity sequence for alkali metals of Na(+)>Li(+)>K(+)≫Rb(+)>Cs(+). Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li(+), K(+), Rb(+), and Cs(+). For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K(+), Rb(+), and Cs(+), suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a.