Cerebellar granule cell cultures of rat express only muscarinic m2 and m3 receptor subtypes and exhibit the pharmacological profile of muscarinic m2 receptors that couple to guanine nucleotide binding proteins to inhibit adenylyl cyclase. In vivo pretreatment with muscarinic agonists desensitizes the muscarinic m2 receptor with 50% complete desensitization within 15 to 20 min. After a 1-hr pretreatment with a maximal concentration of carbachol (short-term desensitization), m2 receptor responsiveness reappeared after a 1-hr treatment of cultures with atropine. However, after a 6-hr pretreatment with carbachol (long-term desensitization), m2 receptor responsiveness did not reappear after 1-hr treatment with atropine. Short-term desensitization was homologous for the m2 receptor because treatment of cultures with carbachol did not alter gamma-aminobutyric acidB receptor-mediated inhibition of adenylyl cyclase. Muscarinic m2 receptor desensitization was not mimicked by the addition of analogs of cyclic AMP, cyclic GMP or diacylglycerol to the cultures. The agonist-induced desensitization was not blocked by a cyclic AMP analog, 8-(4-chlorophenylthio)-cyclic AMP. Pretreatment with antisense oligodeoxynucleotides against the mRNA-encoding beta adrenergic receptor kinase attenuated the desensitization by carbachol (100 microM, 1 hr) of m2 receptors. Irreversible labeling of muscarinic m2 and m3 receptors with [3H]propylbenzilycholine mustard followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a loss of the muscarinic m2 receptor (66-kDa protein), but not the muscarinic m3 receptor (92-kDa protein). We suspect that the short-term desensitization results from the phosphorylation of the muscarinic m2 receptor followed by loss of receptor from the plasma membrane.