Cyclic nucleotide-gated channels contain four ligand-binding subunits, and they are directly activated by the binding of cGMP or cAMP. Channels with different combinations of subunits are known to have different sensitivities to the two nucleotides. However, the consequences of mixed occupancy by cGMP and cAMP are not well understood, and may have important implications for understanding the functions of these channels in different cell types. We studied the activation of homomeric and heteromeric retinal rod cyclic nucleotide-gated channels with the four ligand-binding sites occupied by different combinations of cGMP (a strong agonist) and cAMP (a weak agonist). Control of occupancy was obtained by covalently tethering different numbers of cGMP moieties using the photoaffinity analogue 8-p-azidophenacylthio-cGMP; the remaining sites were then saturated with cAMP, or cGMP, for comparison. The fractional current activated by cAMP increased dramatically as the number of tethered cGMP moieties increased. In homomeric channels comprised of the alpha subunit, cAMP became an effective agonist only after three of the four sites were occupied by tethered cGMP moieties. In contrast, in heteromeric channels comprised of two alpha and two beta subunits, cAMP caused significant activation after two sites were occupied by tethered cGMP moieties. In agreement with earlier work, a single residue on the beta subunit, N1201, accounted for much of the increased efficacy of cAMP on heteromeric channels. The results are consistent with significant interactions between subunits, including the two types of subunits in heteromeric channels.