1. To determine the mechanisms by which cation- or anion-specific channels select between these ions, we have examined the role of electrostatic factors in a typical ligand-gated ion channel, the 5-hydroxytryptamine3 (5-HT3) receptor, by removal and/or insertion of rings of conserved charged residues at either end of the pore. 2. Neutralization of the negatively charged ring at the intracellular end of the channel (E-1'A) results in a nonselective channel (PNa/PCl=0.89). 3. Insertion of positively charged residues at the extracellular end of the pore either results in a nonfunctional receptor (A24'K) or one that remains cation-selective (PNa/PCl=110; S19'R). 4. Combining the removal of a negatively charged ring (E-1'A) with the insertion of a positively charged ring (S19'R), however, results in a channel that is predominantly anion-selective (PNa/PCl=0.37). 5. The data suggest that for the cation-selective 5-HT3 receptor, the control of selectivity exerted by charged rings at either end of the pore is dominated by the ring of negatively charged residues at the intracellular side of the channel. As changing the charge at this position has also been shown to change ionic selectivity in anion-selective receptors, these data suggest that electrostatic factors can control selectivity in the whole Cys-loop family.