Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca2+/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca2+/calmodulin-dependent kinase II such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca2+/calmodulin-dependent kinase KII activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca2+/calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca2+/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.