Selective permeability is a fundamental property of ion channels. The Cys-loop receptor superfamily is composed of both excitatory (ACh, 5-HT) and inhibitory (GABA, glycine) neurotransmitter-operated ion channels. In the GABA receptor, it has been previously shown that the charge selectivity of the integral pore can be altered by a single mutation near the intracellular end of the second transmembrane-spanning domain (TM2). We have extended these findings and now show that charge selectivity of the anionic rho1 GABA receptor can be influenced by the introduction of glutamates, one at a time, over an 8-amino acid stretch (-2' to 5') in the proposed intracellular end of TM2 and the TM1-TM2 intracellular linker. Depending on the position, glutamate substitutions in this region produced sodium to chloride permeability ratios (P(Na)+(/Cl)-) varying from 0.64 to 3.4 (wild type P(Na)+(/Cl)- = 0). In addition to providing insight into the mechanism of ion selectivity, this functional evidence supports a model proposed for the homologous nicotinic acetylcholine receptor in which regions of the protein, in addition to TM2, form the ion pathway.