Cyclic nucleotide-gated (CNG) ion channels play a central role in the signal transduction of olfactory neurons. This channel contains two CNGA2 subunits, one CNGA4 subunit, and one CNGB1b subunit. When heterologously expressed, only the CNGA2 subunits can form functional homotetrameric channels. Question: The purpose of this study is to characterize the molecular processes that are induced by the binding of the first ligand molecule. Methods: CNGA2 tetrameric concatemers containing 0, 1, 2 or 3 disabled subunits (A2-A2-A2-A2, RE-A2-A2-A2, RE-RE-A2-A2, RE-RE-RE-A2) (RE means R538E) were expressed in Xenopus oocytes and analyzed in excised patches by monitoring the ligand-induced current over a wide range of cGMP concentrations. In addition, the kinetics of the only binding event were studied in RE-RE-RE-A2 by means of patch-clamp fluorometry and jumps of a fluorescent cGMP derivative (fcGMP). Results: The concatemers containing 1, 2, and 3 disabled subunits showed a progressive loss of the cGMP sensitivity (EC₅₀) associated with a systematic decrease of the Hill-coefficient, suggesting decreased cooperativity between the remaining functional subunits. When systematically changing the position of the functional subunit in concatemers with only one mutated subunit, the functional parameters were basically similar, though small differences indicate that the position of the free N-terminus and C-terminus with respect to the functional subunit has minor specific effects. In both, macroscopic- and single-channel-experiments, the contribution of one and only one subunit to the activation gating of the whole channel was larger than 40%. The single-channel amplitude of RE-RE-RE-A2 was 4.5 pA, which is similar to that in A2-A2-A2-A2. Conclusions: Our results suggest that in CNGA2 channels the binding of the first ligand is very fast (~10 ms) and that it evokes the opening of the whole tetrameric channel already with the maximum conductance.