G protein-coupled receptors (GPCRs) are subject to the regulation by protein kinases. By controlling the phosphorylation-dephosphorylation balance, protein kinases actively modify GPCR expression and function. In a recent study, we have identified a novel phosphorylation-dependent regulation of Gαi/o-coupled muscarinic acetylcholine receptors. A synapse-enriched protein kinase, Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIα), binds directly and selectively to second intracellular loops of muscarinic M4 receptors (M4Rs). This Ca(2+)-sensitive binding enables CaMKIIα to phosphorylate M4Rs at a selective threonine residue. In rat striatal neurons which abundantly express M4Rs, rapid cytoplasmic Ca(2+) rises enhance the association of CaMKIIα with M4Rs and increase threonine phosphorylation of the receptor. This CaMKIIα-mediated phosphorylation results in a potentiation of M4R activity, which is critical for controlling cellular and behavioral responsivity to dopamine stimulation. In sum, our data identify a novel kinase-GPCR interaction. Through a Ca(2+)/activity-sensitive manner, CaMKIIα contributes to maintaining acetylcholine-dopamine homeostasis in the basal ganglia.