Muscarinic acetylcholine receptors bind allosteric modulators at a site apart from the orthosteric site used by conventional ligands. We tested in cardiac tissue whether modulator binding to ligand-occupied muscarinic M2 receptors is a preferential event that can be detected using a radioactive allosteric agent. The newly synthesized dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) has a particular high potency at M2 receptors occupied by the conventional antagonist N-methylscopolamine (NMS); dissociation of [3H]NMS is half-maximally retarded at an EC50,diss value of 3 nM. Using obidoxime as an "allosteric antagonist," evidence was found that dimethyl-W84 interacts with the postulated common allosteric site. Binding of [3H]dimethyl-W84 (0.3 nM; specific activity, 168 Ci/mmol) was measured in porcine heart homogenates (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4, 23 degrees) in the presence of 1 microM NMS. Homologous competition experiments revealed two components of saturable radioligand binding: one with a high affinity (KD = 2 nM) and small capacity ( approximately 30% of total saturable binding) and the other with a 20,000-fold lower affinity. The Bmax value of the high affinity sites (68 fmol/mg protein) matched muscarinic receptor density as determined by [3H]NMS (79 fmol/mg). Prototype allosteric agents, alcuronium, W84 (the parent compound of the radioligand), and gallamine, displaced high affinity [3H]dimethyl-W84 binding concentration-dependently (pKi values = 8.62, 7.83, and 6.72, respectively). The binding affinities of the modulators were in excellent correlation with their potencies to allosterically stabilize NMS/receptor complexes (EC50,diss = 8.40, 7.72, and 6.74, respectively). We conclude that high affinity binding of [3H]dimethyl-W84 reflects occupation of the common allosteric site of M2 receptors.