The determinants of single channel conductance (gamma) and ion selectivity within eukaryotic pentameric ligand-gated ion channels have traditionally been ascribed to amino acid residues within the second transmembrane domain and flanking sequences of their component subunits. However, recent evidence suggests that gamma is additionally controlled by residues within the intracellular and extracellular domains. We examined the influence of two anionic residues (Asp(113) and Asp(127)) within the extracellular vestibule of a high conductance human mutant 5-hydroxytryptamine type-3A (5-HT(3)A) receptor (5-HT(3)A(QDA)) upon gamma, modulation of the latter by extracellular Ca2+, and the permeability of Ca2+ with respect to Cs+ (P-Ca/P-Cs). Mutations neutralizing (Asp -> Asn), or reversing (Asp -> Lys), charge at the 113 locus decreased inward gamma by 46 and 58%, respectively, but outward currents were unaffected. The D127N mutation decreased inward gamma by 82% and also suppressed outward currents, whereas the D127K mutation caused loss of observable single channel currents. The forgoing mutations, except for D127K, which could not be evaluated, ameliorated suppression of inwardly directed single channel currents by extracellular Ca2+. The P-Ca/P-Cs of 3.8 previously reported for the 5-HT(3)A(QDA) construct was reduced to 0.13 and 0.06 by the D127N and D127K mutations, respectively, with lesser, but clearly significant, effects caused by the D113N (1.04) and D113K (0.60) substitutions. Charge selectivity between monovalent cations and anions (P-Na/P-Cl) was unaffected by any of the mutations examined. The data identify two key residues in the extracellular vestibule of the 5-HT(3)A receptor that markedly influence gamma, P-Ca/P-Cs, and additionally the suppression of gamma by Ca2+.