Abstract Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.