Neonicotinoid insecticides target nicotinic acetylcholine receptors (nAChRs), which, in both vertebrates and invertebrates, mediate fast-acting synaptic neurotransmission in the nervous system. Recently, Kagabu et al. synthesized bis-neonicotinoids. The neural activities of bis-neonicotinoids have been evaluated on the central nerve cord of American cockroaches. However, the action of bis-neonicotinoids on nAChRs expressed by dissociated insect neurons has not yet been studied. Thus, the actions of several alkylene-tethered bis-neonicotinoids on the terminal abdominal ganglion neurons of the American cockroach, Periplaneta americana, were investigated using whole-cell patch-clamp electrophysiology. All of the ligands tested did not induce membrane currents, but reduced the responses to ACh when bath applied prior to co-application with ACh. Of the compounds tested, HK-13, which possesses two imidacloprid units linked with a hexamethylene bridge, had the highest antagonist potency. The antagonist action was reduced, not only by elongating, but also by shortening the linker.