In this study, we characterized a muscarinic acetylcholine receptor (mAChR) potentiator, LY2119620 (3-amino-5-chloro-N-cyclopropyl-4-methyl-6-[2-(4-methylpiperazin-1-yl)-2-oxoethox y]thieno[2,3-b]pyridine-2-carboxamide) as a novel probe of the human M2 and M4 allosteric binding sites. Since the discovery of allosteric binding sites on G protein-coupled receptors, compounds targeting these novel sites have been starting to emerge. For example, LY2033298 (3-amino-5-chloro-6-methoxy-4-methyl-thieno(2,3-b)pyridine-2-carboxylic acid cyclopropylamid) and a derivative of this chemical scaffold, VU152100 (3-amino-N-(4-methoxybenzyl)-4,6-dimethylthieno[2,3-b]pyridine carboxamide), bind to the human M4 mAChR allosteric pocket. In the current study, we characterized LY2119620, a compound similar in structure to LY2033298 and binds to the same allosteric site on the human M4 mAChRs. However, LY2119620 also binds to an allosteric site on the human M2 subtype. [(3)H]NMS ([(3)H]N-methylscopolamine) binding experiments confirm that LY2119620 does not compete for the orthosteric binding pocket at any of the five muscarinic receptor subtypes. Dissociation kinetic studies using [(3)H]NMS further support that LY2119620 binds allosterically to the M2 and M4 mAChRs and was positively cooperative with muscarinic orthosteric agonists. To probe directly the allosteric sites on M2 and M4, we radiolabeled LY2119620. Cooperativity binding of [(3)H]LY2119620 with mAChR orthosteric agonists detects significant changes in Bmax values with little change in Kd, suggesting a G protein-dependent process. Furthermore, [(3)H]LY2119620 was displaced by compounds of similar chemical structure but not by previously described mAChR allosteric compounds such as gallamine or WIN 62,577 (17-beta-hydroxy-17-alpha-ethynyl-delta-4-androstano[3,2-b]pyrimido[1,2-a]benzimi dazole). Our results therefore demonstrate the development of a radioligand, [(3)H]LY2119620 to probe specifically the human M2 and M4 muscarinic receptor allosteric binding sites.