We have investigated the role of intracellular cytoplasmic sequences in the assembly of the mouse muscle nicotinic acetylcholine receptor (AChR) transiently expressed in COS cells. A chimeric protein in which the region from M1 to M4 of the α subunit was replaced by the corresponding region in the β subunit was unable to support AChR assembly when substituted for the α subunit; a chimeric α subunit containing only the long cytoplasmic loop from the β subunit was likewise inactive. Systematic mutation of short segments of the loop identified a sequence of 17 amino acids near the C-terminal end of the loop for which the β sequence could not be substituted. Each of the inactive chimeric and mutated α subunits bound α-bungarotoxin when expressed alone and formed a heterodimer when expressed with the δ subunit. An α subunit truncated after M1 formed both an αδ heterodimer and an αδβ heterotrimer, demonstrating that the cytoplasmic loop is dispensible for the early steps of assembly. A sequence in the long cytoplasmic loop of the α subunit thus appears to play a role in a late step of AChR assembly.