The effects of two competitive antagonists and two allosteric ligands on the rate of dissociation of [3H]N-methylscopolamine ([3H]NMS) were studied at atrial muscarinic acetylcholine M2 receptors by the technique of "infinite dilution." The dissociation rate for [3H]NMS, initiated by diluting the incubation mixture in a 100-fold volume of buffer, was 0.61 +/- 0.10 min-1. Addition of the competitive antagonists, atropine or NMS, to the dilution medium did not alter the observed [3H]NMS dissociation rate. In contrast, gallamine and the bisquaternary, heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl-ammonium bromide) (C7/3'-phth), produced a concentration-dependent slowing of the dissociation rate of [3H]NMS, with IC50 values of 7.5 microM and 196 nM, respectively. Gallamine exhibited an increased modulatory potency when equilibration with the tissue was allowed prior to dilution. The findings showed that the influence of low concentrations of allosteric modulators on the [3H]NMS dissociation rate may be demonstrated separately from any effects on association rate, and that the contact time with the allosteric ligand may influence the extent of these effects.