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Background and Purpose: Affinity-based, selective orthosteric ligands for the muscarinic acetylcholine receptors (mAChRs) are difficult to develop due to high sequence homology across the five subtypes. Selectivity can also be achieved via the selective activation of a particular subtype or signalling pathway. Promisingly, a prior study identified compounds 6A and 7A as functionally selective and G i biased compounds at the M 2 mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M 2 mAChR., Experimental Approach: G protein activation was measured with the TRUPATH assay in M 2 mAChR FlpIn CHO cells. Activity in downstream signalling pathways was determined using the cAMP CAMYEL BRET sensor and assay of ERK 1/2 phosphorylation., Key Results: M 2 mAChRs coupled to Gɑ i1 , Gɑ oA and Gɑ s , but not Gɑ q , in response to canonical orthosteric agonists. Compounds 6A and 7A did not elicit any G protein activation, cAMP inhibition or stimulation, or ERK 1/2 phosphorylation. Instead, a Schild analysis indicates a competitive, antagonistic interaction of compounds 6A and 7A with ACh in the Gɑ i1 activation assay. Overexpression of the M 2 mAChR may suggest an expression-dependent activation profile of compounds 6A and 7A., Conclusions and Implications: These data confirm that the M 2 mAChR preferentially couples to Gɑ i/o and to a lesser extent to Gɑ s in response to canonical orthosteric ligands. However, this study was not able to detect Gɑ i bias of compounds 6A and 7A, highlighting the importance of cellular background when classifying new ligands., Linked Articles: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)