Activation of photoreceptor and olfactory cyclic nucleotide-gated (CNG) channels involves distinct ligand-binding and channel-gating reactions. To dissociate binding from gating, we identified the first competitive antagonists of CNG channels: specific phosphorothioate derivatives of cAMP and cGMP. We also identified membrane-permeant forms of these molecules that are antagonists and that will be useful for elucidating physiological roles for CNG channels in intact cells. The photoreceptor and olfactory CNG channels determine which of the phosphorothioate derivatives are agonists and which are antagonists based on different structural features of the ligand. The photoreceptor channel uses the nature of the purine ring (adenine vs guanine), whereas the olfactory channel uses the isomeric position of the thiophosphate S atom (Rp vs Sp). Interestingly, the same ligand, Rp-cGMPS, has opposite effects on the two channels, activating the photoreceptor channel and antagonizing the olfactory channel. Because Rp-cGMPS binds to both channels but activates only one, the channels must differ in a protein region that couples binding to gating. Chimeric photoreceptor and olfactory CNG channels reveal that the cytoplasmic C-terminal domain determines whether bound ligand activates the channel successfully. Hence, the C terminus contains not only the cyclic nucleotide-binding site, but also a region that couples ligand binding to channel gating.