Aims: Olfactory cyclic nucleotide-gated (CNG) channels are nonselective cation channels that are opened by the binding of the cyclic nucleotides cAMP and cGMP. They form heterotetrameric complexes consisting of three different types of subunits: CNGA2, CNGA4, CNGB1b. CNGA4 and CNGB1b subunits are not functional when expressed alone, but form functional heterotetramers together with CNGA2 subunits. All three types of subunits have a cyclic nucleotide binding domain in their C-terminus. The purpose of this study was to answer the question whether the activation of the heterotetrameric channels is controlled by ligand binding to the CNGA2 subunits alone or also to the CNGA4 and CNGB1b subunits. Methods: Heterotetrameric channels were expressed in Xenopus oocytes. They were studied in excised patches by monitoring ligand binding and gating under both steady-state and non-steady state conditions. Ligand binding was measured by confocal patch-clamp fluorometry using fluorescent cGMP and cAMP analogues (fcGMP, fcAMP). Results: Similar to the homotetrameric CNGA2 channels, we observed also in heterotetrameric channels a crossover of the concentration-binding and the concentration-activation relationships under steady-state conditions. The binding curve of the heterotetrameric channels closely approximates the binding curve of the homotetrameric CNGA2 channels. In order to study the binding to the beta subunits we used two approaches: (1) We expressed either CNGA4 or CNGB1b alone in oocytes and (2) we coexpressed either CNGA4 or CNGB1b with a mutated CNGA2 subunit that has a significantly decreased sensitivity to cGMP and cAMP. Neither fcGMP nor fcAMP generated significant binding to the CNGA4 and CNGB1b subunits. Conclusion: So far, our results suggest that CNGA2A4B1b channel activation is induced by ligand binding to the two CNGA2 subunits only. Possible contributions of the CNGA4, CNGB1b subunits to the heterotetrameric channel gating are discussed.