Peptides corresponding to portions of loop 2 of snake venom curare-mimetic neurotoxins and to a structurally similar region of rabies virus glycoprotein were synthesized. Interaction of these peptides with purified Torpedo electric organ acetylcholine receptor was tested by measuring their ability to block the binding of 125I-labeled alpha-bungarotoxin to the receptor. In addition, inhibition of alpha-bungarotoxin binding to a 32-residue synthetic peptide corresponding to positions 173-204 of the alpha-subunit was determined. Neurotoxin and glycoprotein peptides corresponding to toxin loop 2 inhibited labeled toxin binding to the receptor with IC50 values comparable to those of nicotine and the competitive antagonist d-tubocurarine and to the alpha-subunit peptides with apparent affinities between those of d-tubocurarine and alpha-cobratoxin. Substitution of neurotoxin residue Arg37, the proposed counterpart of the quaternary ammonium of acetylcholine, with a negatively charged Glu residue reduced the apparent affinity about 10-fold. Peptides containing the neurotoxin invariant residue Trp29 and 10- to 100-fold higher affinities than peptides lacking this residue. These results demonstrate that relatively short synthetic peptides retain some of the binding ability of the native protein from which they are derived, indicating that such peptides are useful in the study of protein-protein interactions. The ability of the peptides to compete alpha-bungarotoxin binding to the receptor with apparent affinities comparable to those of other cholinergic ligands indicates that loop 2 of the neurotoxins and the structurally similar segment of the rabies virus glycoprotein act as recognition sites for the acetylcholine receptor. Invariant toxin residues Arg37 and Trp29 and their viral homologs play important, although not essential, roles in binding, possibly by interaction with complementary anionic and hydrophobic subsites on the acetylcholine receptor. The alpha-subunit peptide most likely contains all of the determinants for binding of the toxin and glycoprotein peptides present on the alpha-subunit, because these peptides bind to the 32-residue alpha-subunit peptide with the same or greater affinity as to the intact subunit.