Pre-stimulation of Chinese hamster ovary (CHO) cells expressing the human m1-muscarinic receptor (CHO-m1 cells) with a maximally effective concentration of the muscarinic agonist methacholine resulted in desensitization of Ins(1,4,5)P3 accumulation, apparent as a ∼ 4-fold shift in the agonist dose–response curve. Agonist-induced desensitization was rapid (detectable by 10 s) and concentration dependent (EC50 = 8.2±2.2 µM) and resulted in a complete loss of receptor reserve for the agonist-stimulated Ins(1,4,5)P3 response. An investigation of the possible mechanisms involved in m1-muscarinic receptor desensitization indicated that agonist-induced receptor internalization, PtdIns-(4,5)P2 depletion or an increased rate of Ins(1,4,5)P3 metabolism were not involved. m1-Muscarinic receptors did, however, undergo rapid agonist-induced phosphorylation with a time course that was consistent with an involvement in receptor desensitization. Characterization studies indicated that the receptor-specific kinase involved was distinct from protein kinase C and other second-messenger-dependent protein kinases. Since previous studies have suggested that the m3-muscarinic receptor subtype undergoes agonist-dependent phosphorylation via casein kinase 1α (CK1α) [Tobin, Totty, Sterlin and Nahorski (1997) J. Biol. Chem. 272, 20844–20849], we examined the ability of m1-muscarinic receptors to be phosphorylated by this kinase. In reconstitution experiments, CK1α was able to phosphorylate purified, soluble m1-muscarinic receptors in an agonist-dependent manner.