Cyclic nucleotide-gated (CNG) channels are non-specific cation channels, which mediate e.g. phototransduction in photoreceptors and chemotransduction in olfactory cells. The direct binding of cyclic nucleotides activates the channels. Native olfactory CNG channels are composed of three types of subunits: CNGA2, CNGA4, CNGB1b in the ratio 2:1:1. To gain better insight into the conformational changes associated with the gating of ion channels, activation of CNGA1 and CNGA2 channels in response to a depolarizing voltage jump at different ligand concentrations and also in response to a ligand concentration jump at a constant voltage was studied. The concentration jump was obtained by flash photolysis of caged cNMPs (cyclic nucleoside monophosphate). For CNGA2 channels we showed that at equal degree of activation, the activation time course of homotetrameric channels was similar with cGMP and cAMP and it was also similar in homo- and heterotetrameric channels with the same cyclic nucleotide. Using chimeric channels constructed between retinal CNGA1 and olfactory CNGA2 channels, it was shown that both transmembrane and intracellular channel regions control the activation time course of the CNG channels. Herein, a kinetic model is suggested, which assumes a highly cooperative binding of three ligands: With both cGMP and cAMP, the binding rate of the first and third binding event is two to three orders of magnitude faster than that of the second binding event. In addition, the three binding steps are significantly faster with cGMP than with cAMP.