To learn about the mechanism of ion charge selectivity by invertebrate glutamate-gated chloride (GluCl) channels, we swapped segments between the GluClβ receptor of Caenorhabditis elegans and the vertebrate cationic α7-acetylcholine receptor and monitored anionic/cationic permeability ratios. Complete conversion of the ion charge selectivity in a set of receptor microchimeras indicates that the selectivity filter of the GluClβ receptor is created by a sequence connecting the first with the second transmembrane segments. A single substitution of a negatively charged residue within this sequence converted the selectivity of the GluClβ receptor's pore from anionic to cationic. Unexpectedly, elimination of the charge of each basic residue of the selectivity filter, one at a time or concomitantly, moderately reduced the PCl/PNa ratios, but the GluClβ receptor's mutants retained high capacity to select Cl- over Na+. These results indicate that, unlike the proposed case of anionic Gly- and γ-aminobutyric acid-gated ion channels, positively charged residues do not play the key role in the selection of ionic charge by the GluClβ receptor. Taken together with measurements of the effective open pore diameter and with structural modeling, the study presented here collectively indicates that in the most constricted part of the open GluClβ receptor's channel, Cl - interacts with backbone amides, where it undergoes partial dehydration necessary for traversing the pore.