Retinal rod cyclic nucleotide-gated channels are composed of alpha and beta subunits. We have explored possible subunit arrangements by expressing tandemly linked dimers of both subunits and examining their responses to three different modulating agents. Channels formed from either alpha-beta or beta-alpha heterodimers had functional properties similar to those formed from coexpressed alpha and beta monomers and to native channels. These results point to an alpha-beta-alpha-beta arrangement. To ensure that heterodimers had not flipped around, we coexpressed alpha-alpha dimers with an excess of either beta monomers or beta-beta dimers. Our data indicate that heteromultimers do not form efficiently in an alpha-alpha-beta-beta arrangement. Thus, we propose that native rod cyclic nucleotide-gated channels are arranged with like subunits diagonally opposed: alpha-beta-alpha-beta.