CNG channels are heterotetrameric ligand-gated channels that are essential for visual and olfactory signal transduction. The olfactory CNGA2 subunits form functional homotetrameric channels that can be activated in a cooperative manner by cAMP or cGMP binding to the cyclic nucleotide-binding domains (CNBD) of each subunit. We were interested to kinetically further dissect the molecular mechanism leading to channel activation upon ligand binding and to channel deactivation upon ligand unbinding. Herein, homotetrameric CNGA2 channels were studied in excised patches of Xenopus oocytes expressing these channels by measuring ligand binding and activation as well as ligand unbinding and deactivation under both steady-state and non steady-state conditions, i.e. by recording the respective time courses. Ligand binding was measured by patch-clamp fluorometry (182 or 277 frames per second) using a fluorescent cGMP (fcGMP) (Biskup et al., Nature, 446, 440-3, 2007) which was applied by a fast piezoelectric system. Surprisingly, the unbinding was concentration dependent: the unbinding from the fully liganded channel was approximately 100 times faster than the unbinding from lowly liganded channels. In contrast, the deactivation was concentration independent. The additional information given by the unbinding and deactivation time courses allowed us to extend the previously determined C4L-model (Biskup et al., Nature, 446, 440-3, 2007). Our model includes a separate deactivation pathway to describe the faster unbinding at saturating ligand concentrations compared to low ligand concentrations. Conclusion: Only when either three or four ligands are bound, the channel adopts an open state which allows for a fast unbinding of all four ligands upon ligand removal in the bath.