Evidence suggests that caveolins, 21-24 kDa cholesterol-binding proteins that generally reside in specialized detergent-resistant membrane microdomains, act as signaling scaffolds. Detergent-resistant membranes isolated from rod outer segments (ROS) have been previously shown to contain the photoreceptor G-protein, transducin. In this report we show, by subcellular fractionation, that caveolin-1 is an authentic component of purified ROS. We demonstrate that caveolin-1 in ROS almost exclusively resides in low-buoyant-density, cholesterol-rich, detergent-resistant membranes that can be disrupted by cholesterol depletion using methyl-beta-cyclodextrin (MCD). Cholesterol depletion was also observed to extract a pool of transducin alpha (Talpha) from ROS membranes. Immunoprecipitation with anti-caveolin-1 revealed the association of Talpha in the absence of Tbetagamma. Treatment of ROS with MCD resulted in a 2-fold decrease in recovery of Talpha in anti-caveolin-1 immunoprecipitates. This interaction was also completely disrupted when ROS were exposed to light in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), a nonhydrolyzable GTP analogue. In addition, caveolin-1/Talpha association in the immune complex was disrupted by a peptide based on the primary sequence of the caveolin-1 scaffolding domain. Finally, we confirm the colocalization of caveolin-1 and Talpha in photoreceptors by immunofluorescence microscopy. These results strongly suggest that the association between Talpha and caveolin-1 occurs in cholesterol-rich, detergent-resistant membranes and is likely to be dependent upon the activation state of Talpha.