Xanomeline is a functionally selective M1/M4 muscarinic acetylcholine receptor agonist that nevertheless binds with high affinity to all five subtypes of muscarinic receptors. A novel mode of interaction of this ligand with the muscarinic M1 receptors characterized by persistent binding and receptor activation after extensive washout has been shown previously. In the present study, using human M 1 and M2 receptors expressed in Chinese hamster ovary cells and [3H]N-methylscopolamine as a tracer, we show that persistent binding of xanomeline also occurs at the M2 receptor with similar affinity as at the M1 receptor (KI = 294 and 296 nM, respectively). However, kinetics of formation of xanomeline wash-resistant binding to M2 receptors was markedly slower than to M1 receptors. Xanomeline was a potent fast-acting full agonist in stimulating guanosine 5′-O-(3-[35S]thio)triphosphate binding at M 1 receptors, whereas at M2 receptors it behaved as a potent partial agonist (40% of carbachol maximal response) only upon preincubation for 1 h. Development of xanomeline agonistic effects at the M 2 receptor was slower than its ability to attenuate carbachol responses. We also demonstrate that xanomeline discriminates better between G protein subtypes at M1 than at M2 receptors. Our data support the notion that xanomeline interacts with multiple sites on the muscarinic receptor, resulting in divergent conformations that exhibit differential effects on ligand binding and receptor activation. These conformations are both time- and concentration-dependent and vary between the M1 and the M2 receptor.