The intracellular Ca(2+) concentration in rod outer segments of vertebrate photoreceptors is controlled by Ca(2+) influx through cGMP-gated channels and by Ca(2+) efflux driven by Na/Ca-K exchangers. Previously, we suggested that channel and exchanger are associated (Bauer, P. J., and Drechsler, M. (1992) J. Physiol. (Lond. ) 451, 109-131). This suggestion has been thoroughly examined using a variety of biochemical approaches. First, we took advantage of the fact that cGMP-gated channels bind calmodulin (CaM). Using CaM affinity chromatographic purification of the channel in 10 mm CHAPS, a significant fraction of exchanger was co-eluted with the channel indicating a binding affinity between channel and exchanger. Binding of channel and exchanger was examined more directly by cross-linking of proteins in the rod outer segment membranes. Activation of the channel with cyclic 8-bromo-GMP lead to exposure of a cysteine, which allowed cross-linking of the channel to the exchanger with the thiol-specific reagent dl-1,4-bismaleimido-2,3-butanediol. Cleavage of the cross-links and electrophoretic analysis indicated that a cross-link between the alpha-subunit of the channel and the exchanger formed. Furthermore, a cross-link between two adjacent alpha-subunits of the channel was found, suggesting that the alpha-subunits of the native channel are dimerized. Further support for an interaction between alpha-subunit and exchanger was obtained by in vitro experiments. Specific binding of the exchanger to the alpha-subunit but not to the beta-subunit of the channel was observed in Western blots of purified channel incubated with purified exchanger. This study suggests that two exchanger molecules bind to one cGMP-gated channel and, more specifically, that binding of exchanger molecules occurs at the alpha-subunits, which in the native channel are dimerized. The implications of these findings regarding the possibility of local Ca(2+) signaling in vertebrate photoreceptors will be discussed.