Muscarinic M2 acetylcholine receptors contain an allosteric site that is probably located at the entrance of the ligand binding pocket above the orthosteric binding site. With the orthosteric area not occupied, allosteric agents might gain access to this site. The interaction of allosteric agents with orthoster-occupied receptors is known to depend on the buffer conditions in an alloster-specific fashion. Utilizing the buffer-dependent potency shift as an indicator, we aimed to find out for two rod-like shaped and flexible allosteric agents whether or not there is evidence for a switch in the site of attachment in free compared with [3H]N-methylscopolamine ([3H]NMS)-occupied porcine heart M2 receptors. These agents are the bispyridinium compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl] propane dibromide) and Duo3 (4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype allosteric agents gallamine and alcuronium were included. Inhibition of [3H]NMS association was taken to reflect alloster interaction with free receptors, inhibition of [3H]NMS dissociation indicated binding to [3H]NMS-occupied receptors. In Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degrees C) compared with Mg,Tris,Cl,Pi buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degrees C) WDuo3 underwent the same loss of potency for the interaction with either free or [3H]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentrations of the modulator leading to a half-maximal delay of [3H]NMS association or dissociation, respectively, in Mg,Tris,Cl,Pi compared with Na,K,Pi. For WDuo3 the ratios were PRass=27 and PRdiss=22, respectively. For Duo3, the interaction with free and [3H]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast to the other agents, the concentration-effect curves of which had slope factors nH not different from unity, the curves of Duo3 were steep (nH about -1.6). For alcuronium the shift factors amounted to PRass=29 and PRdiss=25, for gallamine to PRass=216 and PRdiss=159. In conclusion, there was a wide variation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction with either free or [3H]NMS-occupied receptors was always characterized by the same buffer-dependent shift. Thus, even the applied rod-shaped allosteric agents do not appear to switch to the orthosteric site in free compared with orthoster-occupied M2 receptors.