Cyclic nucleotide-gated (CNG) channels are heterotetramers composed of homologue subunits of which only the CNGA2 subunit can form functional homotetrameric channels upon heterologous expression. Because the Hill-coefficient for activation of CNG channels is larger than 1, the activation process must be cooperative. It is not clear whether the cooperativity is generated by the ligand binding, the gating, or both. The activation of CNGA2 channels was studied in excised patches by measuring binding and gating under steady-state and gating under non-steady state conditions. Ligand binding was measured by patch-clamp fluorometry using a fluorescent cGMP analogue (fcGMP). The normalized steady-state binding was larger than the normalized steady-state activation at low [fcGMP] whereas it was smaller at high [fcGMP]. Activation time courses were investigated by cGMP jumps following flash photolysis of a caged cGMP. Global analysis of all data favours a kinetic model with four binding steps and interaction between the binding sites. The first ligand binding activates the channel only partially (Po=0.04), the second is most critical because it drives Po already close to 1. The third and fourth ligand binding further stabilize the open channel. In conclusion, in a homotetrameric CNGA2 channel the binding of only two ligands suffices to exert full channel activation.