Phosphorylation of rhodopsin critically controls the visual transduction cascade by uncoupling it from the G-protein transducin. The kinase primarily responsible for this phosphorylation is rhodopsin kinase, a substrate-regulated kinase that phosphorylates light-activated rhodopsin. Protein kinase C has been implicated in controlling the phosphorylation of both light-activated and dark-adapted rhodopsin. Two of the major rhodopsin phosphorylation sites in vivo, Ser(334) and Ser(338), are effective protein kinase C phosphorylation sites in vitro, while the latter is preferentially phosphorylated by rhodopsin kinase in vitro. Using phosphospecific antibodies against each of these two sites, we show that both sites are under differential spatial and temporal regulation. Exposure of mice to light results in rapid phosphorylation of Ser(338) that is evenly distributed along the rod outer segment. Phosphorylation of Ser(334) is considerably slower, begins at the base of the rod outer segment, and spreads to the top of the photoreceptor over time. In addition, we show that phosphorylation of both sites is abolished in rhodopsin kinase(-/-) mice, revealing an absolute requirement for rhodopsin kinase to phosphorylate rhodopsin. This requirement may reflect the need for priming phosphorylations at rhodopsin kinase sites allowing for subsequent phosphorylation by protein kinase C at Ser(334). In this regard, treatment of mouse retinas with phorbol esters results in a 4-fold increase in phosphorylation on Ser(334), with no significant effect on the phosphorylation of Ser(338). Our results are consistent with light triggering rapid priming phosphorylations of rhodopsin by rhodopsin kinase, followed by a slower phosphorylation on Ser(334), which is regulated by protein kinase C.