Cyclic nucleotide-gated ion channels of retinal photoreceptors and olfactory neurons are differentially activated by ligands that vary only in their purine ring structure. The nucleotide selectivity of the bovine rod cyclic nucleotide-gated channel (cGMP> clMP>> cAMP) was significantly altered by neutralization of a single aspartic acid residue in the binding domain (cGMP/> cAMP> clMP). Substitution by a nonpolar residue at this position inverted agonist selectivity (cAMP>> clMP t> cGMP). These effects resulted from an alteration in the relative ability of the agonists to promote the allosteric conformational change associated with channel activation, not from a modification in their initial binding affinity. We propose a general mechanism for guanine nucleotide discrimination, in common with that observed in high affinity GTP-binding proteins, involving the formation of a pair of hydrogen bonds between the aspartic acid side chain and N1 and N2 of the guanine ring.