The pentametric assembly of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as a ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits, and their ligand specificity characteristics. The receptor from mammalian muscle, with its circular order of homologous subunits (αγαδβ), assembles in a unique arrangement. The residues governing assembly can be ascertained through mutagenesis. Selectivity of certain natural toxins is sufficient to distinguish between sites at the αγ and αδ subunit interfaces. By interchanging residues on the γ and δ subunits through mutagenesis, and ascertaining how they interact with the α subunit, determinants forming the binding sites can be delineated. The α-conotoxins show a 10,000-fold preference for the αδ over αγ subunit interface with αɛ falling in between. The waglerins show a 2,000-fold preference for αɛ over the αγ and αδ interfaces. Finally, the α-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the αγ and αδ interfaces over αɛ. Identification of interactive residues through mutagenesis, when coupled with homology modeling of domains and site-directed residue modification, has revealed important elements of receptor structure.