Ligand binding sites in the muscle nicotinic acetylcholine receptor are generated by pairs of alpha and non-alpha subunits. The non-alpha subunits, gamma, delta and epsilon, contribute significantly to overall affinity of agonists and antagonists, and confer selectivity of these ligands for the two binding sites. By constructing chimeras composed of segments of the various non-alpha subunits and determining ligand selectivity, we have identified four loops, well separated in the linear sequence, that contribute to the non-alpha portion of the binding site. Studies of point mutations in these loops and labeling of engineered cysteines show that the peptide backbones of each non-alpha subunit fold into similar basic scaffolds. Studies of mutations of the peptide antagonists alpha-conotoxin M1 and ImI reveal pairs of residues in the binding site and the toxin that stabilize the complex.