Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M<sub>2</sub>R affinities (p<i>K</i><sub>i</sub> (radioligand competition binding): 9.10-9.59). Binding studies at M<sub>1</sub> and M<sub>3</sub>-M<sub>5</sub> receptors indicated a M<sub>2</sub>R preference. Flow cytometric and high-content imaging saturation and competition binding (M<sub>1</sub>R, M<sub>2</sub>R, and M<sub>4</sub>R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM<sub>2</sub>R cells). Results from dissociation and saturation binding experiments (M<sub>2</sub>R) in the presence of allosteric M<sub>2</sub>R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M<sub>2</sub>R in imaging and binding studies and suggest that they have a dualsteric mode of action.