Rhodopsin is an important member of the superfamily of G protein-coupled receptors. In vitro studies have suggested that multiphosphorylation of rhodopsin is a pivotal step in phototransduction. Because the in vitro biochemical experiments were conducted under nonphysiological conditions, we investigated the phosphorylation of mouse rhodopsin in vivo and determined the sites of phosphorylation and the time course of dephosphorylation. We found that a single phosphate group is incorporated into the rhodopsin molecule in a light-dependent manner, primarily at Ser338 after flashes and at Ser334 after continuous illumination. Dephosphorylation of these sites had different kinetics and spatial distribution in rod outer segments. Dephosphorylation of Ser338 was complete within 30 min, while Ser334 was dephosphorylated much slower (requiring up to 60 min), correlating with the regeneration of rhodopsin. These results suggest that phosphorylation of Ser338 and Ser334 plays different roles in phototransduction.