Pilocarpine (1), a secondary metabolite of several Pilocarpus species, is a therapeutically used partial agonist of muscarinic acetylcholine receptors (mAChRs). The available pharmacological data and structure-activity relationships do not provide comparable data for all five receptor subtypes. In this study, pilocarpine (1), its epimer isopilocarpine (2), racemic analogues pilosinine (3) and desmethyl pilosinine (4), and the respective hybrid ligands with a naphmethonium fragment (5-C6 to 8-C6) were synthesized and analyzed in mini-G nano-BRET assays at the five mAChRs. In line with earlier studies, pilocarpine was the most active compound among the orthosteric ligands 1-4. Computational docking of pilocarpine and isopilocarpine to the active M(2) receptor suggests that the trans-configuration of isopilocarpine leads to a loss of the hydrogen bond from the lactone carbonyl to N(6.52), explaining the lower activity of isopilocarpine. Hybrid formation of pilocarpine (1) and isopilocarpine (2) led to an inverted activity rank, with the trans-configured isopilocarpine hybrid (6-C6) being more active. The hydrogen bond of interest is formed by the isopilocarpine hybrid (6-C6) but not by the pilocarpine hybrid (5-C6). Hybridization thus leads to a modified binding mode of the orthosteric moiety, as the binding mode of the hybrid is dominated by the high-affinity allosteric moiety.