Rhodopsin, the rod cell photoreceptor, undergoes rapid desensitization upon exposure to light, resulting in uncoupling of the receptor from its G protein, transducin (Gt). Phosphorylation of serine and threonine residues located in the COOH terminus of rhodopsin is the first step in this process, followed by the binding of arrestin. In this study, a series of mutants was generated in which these COOH-terminal phosphorylation substrate sites were substituted with alanines. These mutants were expressed in HEK-293 cells and analyzed for their ability to be phosphorylated by rhodopsin kinase and to bind arrestin. The results demonstrate that rhodopsin kinase can efficiently phosphorylate other serine and threonine residues in the absence of the sites reported to be the preferred substrates for rhodopsin kinase. A correlation was observed between the level of rhodopsin phosphorylation and the amount of arrestin binding to these mutants. However, mutants T340A and S343A demonstrated a significant reduction in arrestin binding even though the level of phosphorylation was similar to that of wild-type rhodopsin. Substitution of Thr-340 and Ser-343 with glutamic acid residues (T340E and S343E, respectively) was not sufficient to promote the binding of arrestin in the absence of phosphorylation by rhodopsin kinase. When S343E was phosphorylated, its ability to bind arrestin was similar to that of wild-type rhodopsin. Surprisingly, arrestin binding to phosphorylated T340E did not increase to the level observed for wild-type rhodopsin. These results suggest that 2 amino acids, Thr-340 and Ser-343, play important but distinct roles in promoting the binding of arrestin to rhodopsin.