Background and PurposeAffinity‐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 M₂ mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M₂ mAChR.Experimental ApproachG protein activation was measured with the TRUPATH assay in M₂ 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 ResultsM₂ 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 M₂ mAChR may suggest an expression‐dependent activation profile of compounds 6A and 7A.Conclusions and ImplicationsThese data confirm that the M₂ 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.