Compared to other M 1 muscarinic acetylcholine receptor (M 1 mAChR) agonists, xanomeline demonstrates both reversible and persistent modes of binding to the receptor. In our study, we investigated the long-term consequences of brief incubation of Chinese hamster ovary cells expressing M 1 mAChR (M 1 -CHO) with low concentrations of xanomeline followed by washing off the free drug. Thus, M 1 -CHO cells were exposed to 100 nM xanomeline for 1 h then washed extensively. Washed cells were either used immediately for binding assays or incubated for 23 h in the absence of free xanomeline. Only the latter treatment conditions resulted in marked attenuation of binding of the muscarinic radioligand [ 3 H]N-methylscopolamine ([ 3 H]NMS) to intact cells. Shortening the xanomeline pretreatment period to 1 min had the same trends as the 1 h pretreatment, implying that xanomeline binds instantly to the receptor to elicit long-term wash-resistant effects. Presence of atropine during the brief period of xanomeline pretreatment did not markedly modulate xanomeline's long-term effects, which suggests that persistent anchoring of the xanomeline molecule to the M1 receptor takes place at a site distinct from the orthosteric binding domain. Our findings suggest the possibility of a time-dependent transition of the conformation of the muscarinic M 1 receptor-xanomeline complex between states that vary in their ability to bind [ 3 H]NMS. However, possible involvement of other mechanisms of long-term receptor regulation cannot be discounted. Keywords xanomeline; wash-resistant binding; muscarinic receptors; allosteric modulationThe muscarinic acetylcholine receptor family consists of five receptor subtypes (M 1 -M 5 ) and is part of the superfamily of G-protein coupled receptors [6]. Targeting the M 1 muscarinic acetylcholine receptor (M 1 mAChR) has become of interest due to its role in learning and memory, and the potential it may have in treating certain neurodegenerative diseases [10]. The search for a selective M 1 agonist is necessary to prevent activation of other subtypes such as the M 3, which is responsible for many of the gastrointestinal side effects seen in acetylcholine replacement therapies. Because the orthosteric binding domain on muscarinic receptors where acetylcholine and conventional agonists interact is highly conserved among the five muscarinic receptor subtypes, developing a highly selective M 1 mAChR agonist has been difficult. However, multiple allosteric binding sites have been identified on the M 1 mAChR [4]. Being