[en] The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured using radioimmunoassay. The molar ratio between rhodopsin and total CaM in ROS is 800:1. In the absence of Ca²⁺, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca²⁺, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 x 10^-7 M Ca²⁺ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca²⁺-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca²⁺-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca²⁺. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. These data suggest that both the Ca²⁺-independent binding of CaM to rhodopsin and the Ca²⁺-dependent binding of CaM to the M/sub r/ 140,000 protein represent binding of CaM to a site(s) which is (are) exposed only after denaturation. Ca²⁺-dependent CaM binding in the cytoplasmic fraction is also evaluated with the overlay method. These data suggest that CaM and its binding proteins participate in the regulation of Ca²⁺-sensitive processes primarily on the ROS disk membranes