Receptors have well-conserved regions that are recognized and activated by hormones and neurotransmitters. Most drugs bind to these sites and mimic or block the action of the native ligands. Using a high-throughput functional screen, we identified a potent and selective M 1 muscarinic receptor agonist from a novel structural class. Using a series of chimeric receptors, we demonstrated that this ligand activates the receptor through a region that is not conserved among receptor subtypes, explaining its unprecedented selectivity. This region of the receptor is distinct from the conserved region that is recognized by traditional ligands. The finding that receptors for small-molecule transmitters can have multiple, structurally distinct activation sites has broad implications for the study of receptor structure/function and the potential for the discovery of novel ligands with high selectivity.G-protein-coupled receptors that bind monoamine ligands (e.g., serotonin, adrenaline, dopamine, histamine, and acetylcholine) comprise the most intensively studied and exploited receptor family for the development of therapeutic agents by the pharmaceutical industry. The natural ligands for monoamine receptors are believed to bind a highly conserved pocket located deep within the transmembrane (TM)-spanning regions and to mediate receptor activation primarily through TM3, TM5, TM6, and TM7 (Spalding et al., 1994;Baldwin et al., 1997;Gether, 2000;Lu et al., 2001). Of the amino acids in these regions, 74% are identical in all five muscarinic receptor subtypes (Bonner et al., 1988). Potent small-molecule agonists are also believed to bind monoamine receptors through the same highly conserved regions (Strader et al., 1989(Strader et al., , 1991Wess et al., 1991;Page et al., 1995;Spalding et al., 1998;Ward et al., 1999;Allman et al., 2000).The muscarinic M 1 receptor has been targeted for the discovery of therapeutics for Alzheimer's Disease, and several companies have developed M 1 -selective agonists (e.g., Tecle et al., 1998;Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). Many potent compounds came out of these programs, and several were shown to improve cognition in animals (WAY-132983 and CI-1017; Bartolomeo et al., 2000;Weiss et al., 2000) and people (Xanomeline, Bodick et al., 1997). However, many of the compounds also produced classic muscarinic side effects such as sweating, nausea and diarrhea (Bodick et al., 1997, Bartolomeo et al., 2000Thal et al., 2000). In vitro assays have shown that these compounds activate the M 1 , M 3 , M 4 , and M 5 muscarinic receptor subtypes at similar concentrations (Table 1 and Tecle Wood et al., 1999;Bartolomeo et al., 2000;Wienrich et al., 2001). This may be a direct result of the ligands activating the receptors through regions where the amino acid sequence is almost identical. Since drug interactions with nontarget receptor subtypes are often responsible for the unwanted side effects of commercial pharmaceuticals, there is strong motivation to design more selec...