The activation of cyclic nucleotide-gated (CNG) channels is the final step in olfactory and visual transduction. Previously we have shown that, in addition to their activation by cyclic nucleotides, nitric oxide (NO)-generating compounds can directly open olfactory CNG channels through a redox reaction that results in the S-nitrosylation of a free SH group on a cysteine residue. To identify the target site(s) of NO, we have now mutated the four candidate intracellular cysteine residues Cys-460, Cys-484, Cys-520, and Cys-552 of the rat olfactory rCNG2 (alpha) channel into serine residues. All mutant channels continue to be activated by cyclic nucleotides, but only one of them, the C460S mutant channel, exhibited a total loss of NO sensitivity. This result was further supported by a similar lack of NO sensitivity that we found for a natural mutant of this precise cysteine residue, the Drosophila melanogaster CNG channel. Cys-460 is located in the C-linker region of the channel known to be important in channel gating. Kinetic analyses suggested that at least two of these Cys-460 residues on different channel subunits were involved in the activation by NO. Our results show that one single cysteine residue is responsible for NO sensitivity but that several channel subunits need to be activated for channel opening by NO.