Copyright © 2018 American Chemical Society. The muscarinic M 2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 μs in total) to study conformational changes triggered by the inverse agonist R-(-)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M 2 receptor (PBD ID 4MQS) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M 2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY2119620 when bound simultaneously with the orthosteric agonist iperoxo and find that it restricts the conformation of Trp422 7.35 in a position that modulates the orientation of the Tyr426 7.39 at the orthosteric-binding pocket.