Aqueous exposure of critical residues in the selectivity region of voltage gated Na+ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3) and IV (D4) of the human heart Na+ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate (MTSEA+) and (2-sulfonatoethyl)methanethiosulfonate (MTSES-) in all six of the Cys-substituted channels, including Trp --> Cys substitutions at homologous positions in D3 and D4 that were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification, each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >10(2). Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C (the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even though both were exposed. Neither position responded to MTSES- suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES- markedly increased, and MTSEA+ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp1713 acts electrostatically in an unrestricted region. These results suggest that Lys1418 lies in a restricted region favorable to cations, whereas Asp1713 is at a more peripheral location in the Na+ channel pore.