Cyclic nucleotide-gated (CNG) channels mediate phototransduction in photoreceptors and chemotransduction in olfactory neurons. The activation of these channels is governed by the binding of cyclic nucleotides (cGMP, cAMP). Native olfactory CNG channels are heterotetramers of three different subunits, 2 x CNGA2, CNGA4, CNGB1b. In contrast to CNGA2, CNGA4 and CNGB1b subunits do not form functional channels on their own, but modulate the function of the channels in combination with CNGA2. The individual contribution of these subunits to the activation process, in particular their ability to bind cyclic nucleotides, has not been elucidated so far. Heterotetrameric CNGA2:A4:B1b channels were expressed in Xenopus oocytes and 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 a fluorescent cGMP analogue (fcGMP). Similar to the homotetrameric CNGA2 channels, we observed a crossover of the normalized steady-state binding and steady-state activation relationships. Using TFP-labelled CNGA4 and CNGB1b subunits alone, we show that these subunits do reach the plasma membrane in the absence of CNGA2. Further, we coexpressed each of these 2 subunits with either CNGA2(T539M) or CNGA2(R538E), two mutated CNGA2 subunits with disabled binding sites. Under these conditions, we observed significant fcGMP binding to the CNGA4, but not to the CNGB1b subunit. Moreover, a CNGA2 chimera with a CNGA4 binding site produced functional channels whereas a respective construct with a CNGB1b binding site failed. Our results suggest that CNGA2:A4:B1b channels are activated by ligand binding to CNGA2 and CNGA4 subunits, but not to the CNGB1b subunit.