Nicotinic acetylcholine receptors (AChRs) are activated by ACh binding to two sites located on different α subunits. The two α subunits, α_γ and α_δ, are distinguished by their interface with γ and δ subunits. We have characterized the formation of the ACh binding sites and found, contrary to the current model, that the sites form at different times and in a set order. The first site forms on α_γ subunits during the process of subunit assembly. Our data are consistent with the appearance of this site on αβγδ subunit tetramers soon after the site for the competitive antagonist α-bungarotoxin has formed and δ subunits have assembled with αβγ trimers. The second site is located on α_δ subunits and forms after AChR subunits have assembled into α₂βγδ pentamers. By determining the order in which the ACh binding sites form, we have also identified the sites in which the δ and second α subunits associate during subunit assembly.