A series of 6H,13H-pyrazino[1,2-a;4,5-a′]diindole analogs was synthesized in order to probe the pharmacophore hypothesis for allosteric ligands of muscarinic M 2 receptors. The 3D structure of the novel ring system was determined by means of NMR spectroscopy and X-ray diffraction revealing a totally flat geometry. Low binding affinities for the [ 3 H]N-methylscopolamine-occupied M 2 receptors (reflected by EC 50,diss ) indicated that the spatial arrangement of the pharmacophore elements (two aromatic rings flanked by two cationic centers) incorporated in the bisquaternary analogs 5 and 6 is unfavorable for strong ligand–receptor interactions. Due to the structural similarity of the novel compounds to neuromuscular-blocking agents, their affinities (reflected by K i ) to the muscle type of nicotinic acetylcholine receptors were also determined. The dimethyl and diallyl analogs 5 and 6 exhibited rather high affinities to the muscle type of nicotinic acetylcholine receptors, suggesting a pronounced neuromuscular-blocking activity. Compound 5 showed a 34-fold higher affinity for the muscle type nAChR than for the allosteric site of M 2 receptors.