The cGMP-gated cation channel mediating visual transduction in retinal rods was recently found to comprise at least two subunits, 1 and 2 (or alpha and beta). SDS gels of the purified channel show, in addition to a 63-kDa protein band (subunit 1), a 240-kDa protein band that binds Ca(2+)-calmodulin, a modulator of the channel. To examine any connection between subunit 2 and the 240-kDa protein, cGMP-gated channels formed from the expressed cloned subunits in human embryonic kidney (HEK) 293 cells were tested for Ca(2+)-calmodulin effect. Homooligomeric channels formed by subunit 1 alone showed no sensitivity to Ca(2+)-calmodulin, and neither did heterooligomeric channels formed by subunit 1 and the short alternatively spliced form of subunit 2 (2a). By contrast, the cGMP half-activation constant (K1/2) for heterooligomeric channels formed from subunit 1 and the long form of subunit 2 (2b) was increased 1.5- to 2-fold by Ca(2+)-calmodulin, similar to the increase observed for the native channel. In Western blots of rod outer segment membranes, a subunit 2-specific antibody also recognized the 240-kDa protein. Finally, amino acid sequences derived from peptide fragments of the bovine 240-kDa protein showed approximately 80% identity to regions of subunit 2b of the human channel. These results together suggest that subunit 2b of the rod channel is a component of the 240-kDa protein and that it mediates the Ca(2+)-calmodulin modulation of the channel.