Modification of positively charged residues, Lys and Arg, in cobrotoxin revealed that Lys-27, Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 of cobrotoxin were essential for the lethality and binding activity to nicotinic acetylcholine receptor (nAChR). The antigenicity of cobrotoxin was drastically diminished when Lys-47, Arg-28, Arg-30, Arg-33, and Arg-36 were modified, while that of the Lys-27-modified derivative was not significantly changed. The CD spectra of cobrotoxin displayed similar patterns after modification of Lys-27, Lys-47, and Arg-28. These findings suggest that Lys-27, Lys-47, and Arg-28 residues may be related to the direct binding to nAChR, and that there is no involvement of Lys-27 in the antigenic determinants of cobrotoxin. Extending the modification to Arg-30, Arg-33, and Arg-36 caused progressive conformational changes of cobrotoxin and resulted in decreased binding activity to antibody and nAChR. This indicates that Arg-30, Arg-33, and Arg-36 may be of structural importance for maintaining the active conformation of cobrotoxin. These results, together with the facts that Tyr-25, Tyr-35, and Trp-29 of cobrotoxin are important in nAChR binding activity, but Trp-29 and Tyr-35 residues are not essential for the antigenicity, suggest that the structural loop II of cobrotoxin is the main binding region for nAChR and the epitope in that region is conformation-dependent.