Muscarinic acetylcholine receptors contain two distinct ligand binding sites, i.e. the orthosteric site for acetylcholine and other conventional ligands, and an allosteric site located at the entrance of the ligand binding pocket. We used a set of allosteric agents to probe whether muscarinic M2 receptors whose orthosteric site is occupied by an agonist still reveal the common allosteric site that has been identified in M2 receptors being occupied by an orthosteric antagonist (N-methylscopolamine, NMS). Equilibrium and dissociation binding experiments were carried out in porcine heart homogenates using either the agonist [3H]oxotremorine M ([3H]OxoM) or the antagonist [3H]NMS. The affinities of the allosteric agents were determined for the radioligand-occupied receptor states and, additionally, for the radioligand-free (ground state) M2 receptor. The archetypal agent W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide] and its bispyridinio middle chain analogue WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl]propane dibromide) had a clearly lower affinity for [3H]OxoM-liganded receptors compared with [3H]NMS-liganded and ground state receptors. In contrast, a derivative resembling only one half of W84 had equal affinities for both radioligand-occupied receptor states. Also, the agents gallamine and obidoxime did not discriminate between [3H]OxoM- and [3H]NMS-occupied receptors. The allosteric antagonistic tool obidoxime inhibited WDuo3 action in [3H]OxoM-liganded receptors with the same potency as in [3H]NMS-liganded receptors. We conclude that the common allosteric site is still present in OxoM-liganded M2 receptors, but its spatial conformation is considerably altered compared with NMS-liganded receptors.