Objective: Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to an intracellular cyclic nucleotide-binding domain (CNBD). The molecular events translating the binding to the pore opening are still unknown. Methods: We investigated the role of intracellular channel regions on the activation process by constructing chimeric channels in which the N-terminus, the S4-S5 linker, the C- terminus, and the CNBD of the retinal CNGA1 subunit were systematically replaced by respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by an interaction energy according to the principles of thermodynamic double-mutant cycle analysis. Results: We show that all four intracellular regions, including the short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits as determined from the Hill coefficients. The interaction energies are specific for pairs of regions, thereby depending characteristically on the nature of the other intracellular regions. The effect of the S4-S5 linker substitution is coupled both to the N- terminus and to the C-linker effect but not to the CNBD effect. Conclusion: It is concluded that the N-terminus and the S4-S5 linker essentially cooperate with the CNBD and the C-linker in the activation gating of CNG channels.