To explore the role of pore-lining amino acids in Na+ channel ion-selectivity, pore residues were replaced serially with cysteine in cloned rat skeletal muscle Na+ channels. Ionic selectivity was determined by measuring permeability and ionic current ratios of whole-cell currents in Xenopus oocytes. The rSkM1 channels displayed an ionic selectivity sequence Na+ > Li+ > NH4+ > > Cs+ and were impermeable to divalent cations. Replacement of residues in domain IV showed significantly enhanced current and permeability ratios of NH4+ and K+, and negative shifts in the reversal potentials recorded in the presence of external Na+ solutions when compared to cysteine mutants in domains I, II, and III (except K1237C). Mutants in domain IV showed altered selectivity sequences: W1531C (NH4+ > K+ > Na+ > or = Li+ approximately Cs+), D1532C, and G1533C (Na+ > Li+ > or = NH4+ > K+ > Cs+). Conservative replacement of the aromatic residue in domain IV (W1531) with phenylalanine or tyrosine retained Na+ selectivity of the channel while the alanine mutant (W1531A) reduced ion selectivity. A single mutation within the third pore forming region (K1237C) dramatically altered the selectivity sequence of the rSkM1 channel (NH4+ > K+ > Na+ > or = Li+ approximately Cs+) and was permeable to divalent cations having the selectivity sequence Ca2+ > or = Sr2+ > Mg2+ > Ba2+. Sulfhydryl modification of K1237C, W1531C or D1532C with methanethiosulfonate derivatives that introduce a positively charged ammonium group, large trimethylammonium moiety, or a negatively charged sulfonate group within the pore was ineffective in restoring Na+ selectivity to these channels. Selectivity of D1532C mutants could be largely restored by increasing extracellular pH suggesting altering the ionized state at this position influences selectivity. These data suggest that K1237 in domain III and W1531, D1532, and G1533 in domain IV play a critical role in determining the ionic selectivity of the Na+ channel.