Muscarinic receptors of the m2 subtype expressed in Chinese hamster ovary cells were labeled with [methyl-3H]acetylcholine([3H]ACh), and the rate of dissociation in the presence and absence of several compounds known to exert allosteric effects on labeled antagonist binding was observed. At 25 degrees C, [3H]ACh bound to the receptors with a Kd of 1.2 nM and dissociated with a half-time of 1.6 min. This binding was sensitive to appropriate concentrations of guanine nucleotide and the muscarinic antagonist N-methylscopolamine (NMS). Gallamine, tetrahydroaminoacridine, physostigmine, obidoxime, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) all inhibited the binding of [3H]ACh and all slowed the rate of dissociation of [3H]ACh in a concentration-dependent manner. However, the nature of some of the allosteric effects differed from previous studies that used other labeled ligands. In particular, TMB-8, which is very effective in slowing the dissociation of the antagonist [3H]NMS, had much weaker effects on the dissociation of [3H]ACh. Furthermore, TMB-8 was able to partially reverse the stronger effects of gallamine on the dissociation of [3H]ACh, consistent with the possibility that TMB-8 and gallamine share a common site on the receptor. In summary, the binding of ACh to muscarinic receptors is subject to allosteric regulation, and assays using [3H]ACh may be especially useful in the evaluation of potential allosteric regulators of muscarinic systems.