Phosducin is a major phosphoprotein of rod photoreceptors that interacts with the Gbetagamma subunits of heterotrimeric G proteins in its dephosphorylated state. Light promotes dephosphorylation of phosducin; thus, it was proposed that phosducin plays a role in the light adaptation of G protein-mediated visual signaling. Different functions, such as regulation of protein levels and subcellular localization of heterotrimeric G proteins, transcriptional regulation, and modulation of synaptic transmission have also been proposed. Although the molecular basis of phosducin interaction with G proteins is well understood, the physiological significance of light-dependent phosphorylation of phosducin remains largely hypothetical. In this study we quantitatively analyzed light dependence, time course, and subcellular localization of two principal light-regulated phosphorylation sites of phosducin, serine 54 and 71. To obtain physiologically relevant data, our experimental model exploited free-running mice and rats subjected to controlled illumination. We found that in the dark-adapted rods, phosducin phosphorylated at serine 54 is compartmentalized predominantly in the ellipsoid and outer segment compartments. In contrast, phosducin phosphorylated at serine 71 is present in all cellular compartments. The degree of phosducin phosphorylation in the dark appeared to be less than 40%. Dim light within rod operational range triggers massive reversible dephosphorylation of both sites, whereas saturating light dramatically increases phosphorylation of serine 71 in rod outer segment. These results support the role of phosducin in regulating signaling in the rod outer segment compartment and suggest distinct functions for phosphorylation sites 54 and 71.