The muscarinic acetylcholine receptors (mAChRs) comprise a family of G‐protein‐coupled receptors, all of which are expressed in the CNS. Physiological, behavioral, and knock‐out mouse studies all suggest that these receptors play crucial roles in many CNS functions and disorders. Unfortunately, the structures of the five subtypes of mAChRs are very highly conserved in the transmembrane regions where ACh binds; this has hampered the development of subtype‐selective agonists and antagonists. Additionally, the mAChRs also play essential roles in the autonomic system; this means that non‐selective muscarinic agents have serious dose‐limiting side effects. One approach to developing ligands with sufficient selectivity has been to target allosteric sites on the receptors, which are typically much less conserved than the orthosteric site. BQCA is a premier example of a positive allosteric modulator that is highly selective for the M1 mAChR, which is a subtype whose enhancement is likely to benefit cognitive deficits, including those of Alzheimer's Disease. BQCA enhances the potency of ACh by 100‐fold or more at M1, while having no effect at the other subtypes. Such selectivity can be due either to highly selective affinity for M1 or to neutral cooperativities at the other subtypes, and it is of significant interest to determine which mechanism is at work for this prototypical agent. We have used mutagenesis to identify residues that are key to the selective PAM activity. In agreement with others, we found that the aromatic nature of M1Y5.29 is essential (superscript numerals refer to the system of Ballesteros and Weinstein); Y=>A mutation abolishes activity, while Y=>F preserves it. Of the five subtypes, only M5 lacks Y or F at this site. We found that the M5Q=>Y5.29 mutant displays modest PAM activity. Additionally, the PAM activity of the double mutant M1E=>A7.32, E=>A7.36 is markedly attenuated, whereas the M5Y5.29E7.32E7.36 mutant has PAM activity equal to M1. Unlike the case with M5, the M2E7.32E7.36, M3E7.32E7.36, and M4E7.32E7.36 mutants exhibit only moderate PAM activity, despite the Y or F present at position 5.29. Finally, the interactions between BQCA and ACh in inhibiting the binding of the labeled orthosteric antagonist [3H]N‐methylscopolamine to M1 vs M1A7.32A7.36 and M5Y5.29 vs M5Y5.29E7.32E7.36 were evaluated using a mathematical allosteric model. The mutations were found to exert marked changes in the cooperativity between BQCA and ACh, but not to alter the affinity of BQCA for the receptor. This indicates that the region near the top of TM7 that contains E7.32 and E7.36 is not part of the binding site. In agreement with this, BQCA was not found to be competitive with the allosteric modulator W84, which has previously been shown to bind within this region.Support or Funding InformationThis project is funded, in part, under a grant with the Pennsylvania Department of Health using Tobacco CURE Funds. The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions. Also supported by the National Institute on Aging [Grant R01AG005214] and by funds from the Department of Psychiatry, Penn State University College of Medicine