Chinese hamster ovary (CHO) cells transfected with human m3 muscarinic receptor cDNA (Bmax, 1343 +/- 46.8 fmol/mg of protein) were used to investigate agonist-mediated muscarinic receptor desensitization. Stimulation of CHO-m3 cells with a maximal dose of carbachol resulted in a biphasic production of mass inositol-1,4,5-trisphosphate [Ins(1,4,5)P3], measured by radioreceptor binding assay. The first phase comprises a rapid 8-10-fold increase in Ins(1,4,5)P3 that peaks after 10 sec and falls to levels 3-4-fold over basal within 1 min Ins(1,4,5)P3 rises again over the next 20 min to approximately 8-10-fold above basal, where levels are sustained for at least 2 hr. This later phase is, therefore, considered to be a desensitization-resistant component of m3 receptor activation. A 5-min pre-exposure of CHO-m3 cells to carbachol resulted in attenuation of the initial peak Ins(1,4,5)P3 response to a subsequent application of agonist. The attenuation of the Ins(1,4,5)P3 response was reversible with a t1/2 of approximately 7.5 min. Desensitization and recovery of the peak Ins(1,4,5)P3 response correlated with a decrease and subsequent recovery of m3 receptor-mediated mobilization of intracellular calcium stores, suggesting that the consequence of peak Ins(1,4,5)P3 desensitization is a reduced calcium mobilization response. N-[3H]Methylscopolamine binding to intact cells revealed that there was no change in cell surface m3 receptors during the 5-min pre-exposure to agonist, indicating that the mechanism of muscarinic receptor desensitization described here is not sequestration or internalization of receptors.