α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7 nAChR-targeting α-conotoxin ImI, blocked α7 and muscle nAChRs without displacing α-bungarotoxin (Ellison et al. 2003, 2004), suggesting binding at a different site. We synthesized α-conotoxin ImII, its ribbon isomer (ImIIiso), 'mutant' ImII(W10Y) and found similar potencies in blocking human α7 and muscle nAChRs in Xenopus oocytes. Both isomers displaced [125I]-α- bungarotoxin from human α7 nAChRs in the cell line GH4C1(IC5017 and 23 μM, respectively) and from Lymnaea stagnalis and Aplysia californica AChBPs (IC502.0-9.0 μM). According to SPR measurements, both isomers bound to immobilized AChBPs and competed with AChBP for immobilized α-bungarotoxin (Kdand IC502.5-8.2 μM). On Torpedo nAChR, α-conotoxin [125I]-ImII(W10Y) revealed specific binding (Kd1.5-6.1 μM) and could be displaced by α-conotoxin ImII, ImIIiso and ImII(W10Y) with IC502.7, 2.2 and 3.1 μM, respectively. As α-cobratoxin and α-conotoxin ImI displaced [125I]-ImII(W10Y) only at higher concentrations (IC50≥ 90 μM), our results indicate that α-conotoxin ImII and its congeners have an additional binding site on Torpedo nAChR distinct from the site for agonists/competitive antagonists. © 2009 International Society for Neurochemistry.