We have used transient expression in COS cells of the subunits of the nicotinic acetylcholine receptor (AChR) from mouse skeletal muscle to investigate the role of transmembrane and cytoplasmic domains of the delta subunit in assembly of the AChR. When chimeric subunits whose extracellular amino- and carboxyl-terminal domains were from the delta subunit and whose transmembrane and cytoplasmic domains were from either the beta, gamma, or epsilon subunit were expressed with alpha, beta, and epsilon subunits, alpha-bungarotoxin-binding activity appeared on the surface of the transfected cells. The resulting receptor complexes each had sedimentation constants resembling those of the native AChR, consistent with a pentameric structure. Further investigation of the delta beta chimeric subunit showed that it formed a heterodimer with the alpha subunit and that the resulting subunit bound d-tubocurarine with an affinity similar to that of the alpha delta heterodimer; delta beta also formed a heterodimer with a form of the alpha subunit that is truncated after the first transmembrane domain. A heterodimer formed from the epsilon beta and alpha subunits also bound d-tubocurarine with an affinity similar to that of the alpha epsilon heterodimer. When both epsilon beta and delta beta subunits were substituted for the epsilon and delta subunits, respectively, a receptor complex was formed whose structure appeared to be alpha 2 beta(epsilon beta)(delta beta). These results show that, as with the epsilon subunit, the identity of the delta subunit in AChR assembly arises from the extracytoplasmic domains of the subunit.