Increases in the extracellular hydrogen ion concentration ([H+]o) but not the intracellular concentration ([H+]i) antagonized the inward going membrane currents recorded from isolated cone horizontal cells during application of quisqualate, alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid, and kainate. The pK determined from a titration curve was 6.5 with a slope greater than 1, indicating protonation of several histidines. The reduction in membrane current was voltage-independent. The affinity of the agonist for the receptor, the single-channel conductance, and the open time were unaffected by [H+]o. [H+]o antagonism was not the result of charge neutralization such as screening surface charge. Diethylpyrocarbonate, a histidine-modifying reagent, reduced the agonist-induced current, but disulfide- and sulfhydryl-modifying reagents were ineffective. These results suggest that histidine groups on the external face of the channel protein provide a functional site regulating channel gating.