Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a beta roll followed by two alpha helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.