The cytoplasmic loops of rhodopsin, the rod cell photoreceptor, play important regulatory roles in the activation of both rhodopsin kinase and the rod cell G protein, Gt. A number of studies have identified domains in rhodopsin that are important for the activation of Gt. However, less is known concerning the cytoplasmic regions that regulate phosphorylation of the photoreceptor by rhodopsin kinase. To identify regions that participate in these processes, a series of alanine mutations were generated in the three cytoplasmic loops of rhodopsin and transiently expressed in HEK-293 cells. Membranes prepared from these cells were reconstituted with the opsin chromophore, 11-cis-retinal, and characterized for their ability to undergo light-dependent phosphorylation by rhodopsin kinase and to catalyze GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) binding to Gt. We have identified mutants that fall into three distinct categories: 1) those that show altered phosphorylation but normal Gt activation, such as T62A/V63A/Q64A and R147A/F148A/G149A in Loops I and II, respectively; 2) mutants that have reduced ability to activate Gt but are phosphorylated normally, including T242A/T243A and V250A/T251A/R252A in Loop III; and 3) mutants that affect both phosphorylation and Gt activation, including A233G/A234G/A235G and A233N/A234N/A235N in Loop III. The use of these two assays in parallel have allowed us to distinguish the presence of distinct functional domains within the cytoplasmic loops which are specific for interaction with rhodopsin kinase or Gt.