The functional consequences of single proton transfers occurring in the pore of a cyclic nucleotide-gated channel were observed with patch recording techniques. These results led to three conclusions about the chemical nature of ion binding sites in the conduction pathway : The channel contains two identical titratable sites, even though there are more than two (probably four) identical subunits; the sites are formed by glutamate residues that have a pK(a) (where K-a is the acid constant) of 7.6; and protonation of one site does not perturb the pK(a) of the other. These properties point to an unusual arrangement of carboxyl side-chain residues in the pore of a cation channel.