The effects of prolonged exposure of M(2) muscarinic acetylcholine receptors (mAChRs), stably expressed in Chinese hamster ovary cells, to the allosteric modulators gallamine, alcuronium, and heptane-1,7-bis (dimethyl-3'-phthalimidopropyl)-ammonium bromide (C(7)/3'-phth) were compared with the effects of the agonist carbachol (CCh) and antagonists atropine and N-methylscopolamine (NMS). Intact cell saturation binding assays using [(3)H]NMS found that pretreatment of the cells for 24 h with CCh caused a significant down-regulation of receptor number, whereas atropine, NMS, and all three allosteric modulators caused receptor up-regulation. Functional assays using a cytosensor microphysiometer to measure whole-cell metabolic rate found no acute effects of gallamine on receptor signaling, whereas atropine seemed to behave as an inverse agonist. Pretreatment of the cells with gallamine (20 microM) or atropine (20 nM) resulted in a significant enhancement of the maximal effect evoked by CCh. In contrast, CCh (100 microM) pretreatment resulted in a significant reduction in maximal receptor signaling capacity. Time-course experiments revealed that the effects of atropine and gallamine on receptor up-regulation are only visualized after at least 12-h ligand exposure, compared with the more rapid effects of CCh, which achieve steady-state down-regulation within 90 min. Additional experiments monitoring CCh-mediated M(2) mAChR internalization in the presence of gallamine revealed that part of the mechanism underlying the effects of the modulator on receptor expression may involve a change in receptor internalization properties. These findings suggest that, like orthosteric ligands, G protein-coupled receptor allosteric modulators also are able to mediate long-term effects on receptor regulation.