A set of seven peptides constituting the various loops and most of the surface areas of alpha-bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bind Torpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1-16, 26-41, and 45-59, were able to bind 125I-labeled AChR and, conversely, 125I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28, Lys-26 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1-16, 26-41, and 45-59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45-47).