The gamma subunit of rod-specific cGMP phosphodiesterase 6 (PDE6gamma), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6gamma on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6gamma may increase or decrease the ability of PDE6gamma to deactivate phototransduction. To resolve role of phosphorylation of PDE6gamma in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6gamma (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6gamma, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6gamma is essential for the regulation of G-protein signaling.