Cyclic nucleotide-gated (CNG) channels play a role in olfactory transduction. Olfactory CNG channels consist of three different subunits, 2XCNGA2, 1XCNGA4 and 1XCNGB1b. Only the CNGA2 subunits can form functional homotetrameric channels. It is still unclear how ligand binding is translated to channel gating in the homotetrameric CNGA2 channels. Therefore, we characterized the work of only one CNGA2 subunit in homotetrameric CNGA2 channels. We used the concatenation technique in combination with the patch-clamp technique. Selected subunits were rendered almost insensitive to cGMP by a point mutation (R538E) in the binding domain. First, we show direct evidence indicating that the concatenation technique does not have any effect on channel function: 1) The concentration-response relationship of wt-wt-wt-wt concatemer was almost identical to that of channels formed by non-concatenated subunits (4wt). 2) When coexpressing wt-wt-wt-wt concatemer with mut subunits, the concentration-response relationship was indistinguishable from that of wt-wt-wt-wt concatemer alone. 3) The amplitude of the single-channel current and the open probability of wt-wt-wt-wt channels were not significantly different from that of 4wt channels in the presence of saturating cGMP concentrations. Then, the concentration-response relationships obtained from the concatemers containing 1, 2, 3 or 4 mutated subunits showed that an increase of the number of mut subunits in the concatemeric channels caused graded losses in cGMP sensitivity (EC50) associated with a systematic decrease of the Hill coefficient (H). Moreover, we showed that the position of the functional subunit(s) in the concatemers is irrelevant for the channel function. We further characterized the CNGA2-channel activation induced by the first ligand binding step in mut-mut-mut-wt channels by means of the single-channel recordings and a global fit analysis with Markovian models. We found that the first binding step is sufficient to evoke significant channel opening to the full conductance level. The second binding step is the most important step because this step drives the channel from the closed to the maximal open state. Together, these results provide direct information about the functional role of individual subunits in homotetrameric CNGA2 channels.