Cyclic nucleotide-gated (CNG) channels are directly activated by the binding of several ligands. For these channels as well as for other allosteric proteins, the functional effects of each ligand-binding event have been difficult to assess because ligands continuously bind and unbind at each site. Furthermore, in retinal rod photoreceptors the low cytoplasmic concentration of cyclic GMP means that channels exist primarily in partially liganded states, so it is important to determine how such channels behave. Previous studies of single channels have suggested that they occasionally open to subconducting states at low cGMP, but the significance of these states and how they arise is poorly understood. Here we combine the high resolution of single-channel recording with the use of a photoaffinity analogue of cGMP that tethers cGMP moieties covalently to their binding sites to show single retinal CNG channels can be effectively locked in four distinct ligand-bound states. Our results indicate that channels open more than they would spontaneously when two ligands are bound (approximately 1% of the maximum current), significantly more with three ligands bound (approximately 33%), and open maximally with four ligands bound. In each ligand-bound state, channels opened to two or three different conductance states. These findings place strong constraints on the activation mechanism of CNG channels.