Comparative structure-function studies have been carried out for α-conotoxin GI acting on nicotinic acetylcholine receptors (AChR) from mammalian muscles and from the electric organ of the Torpedo californica ray and for α-conotoxin ImI, which targets the neuronal α7 AChR. A series of analogs has been prepared for this purpose: chemically modified derivatives, including a covalently linked dimer of GI, as well as analogs wherein one or several amino acid residues have been changed using solid-phase peptide synthesis. The activity of all compounds was assessed in competition with radioiodinated and/or tritiated α-conotoxin GI for binding to the membrane-bound AChR of Torpedo californica. Binding of radioiodinated α-conotoxin GI dimer was also monitored directly, revealing the largest, as compared to all other analogues, difference in the affinity between the two binding sites in the receptor (KD∼ 11 and 1200 nM). Comparison of binding data with the results of CD measurements point to important role of the spatial organization of the α-conotoxin second loop in manifestation of their "muscle" or "neuronal" specificity.