Through the linkage of two muscarinergic M3 receptor ligands to fluorescent tetramethylrhodamine- and cyanine-5-type dyes, two novel tool compounds, OFH5503 and OFH611, have been developed. Based on the suitable binding properties and kinetics related to the M3 subtype, both ligand-dye conjugates were found to be useful tools to determine binding affinities via flow cytometric measurements. In addition, confocal microscopy underlined the comparably low unspecific binding and the applicability for studying M3 receptor expression in cells. Along with the proven usefulness regarding studies on the M3 subtype, the conjugates OFH5503 and OFH611 could, due to their high affinity to the M1 receptor, evolve as even more versatile tools in the field of research on muscarinergic receptors.