The 43 kDa protein, a cytoplasmic peripheral membrane protein, is closely associated with the acetylcholine receptor (AChR) at the neuromuscular junction, where it is thought to anchor the receptor in the postsynaptic membrane. We have used the 43 kDa protein-induced clustering of AChRs that occurs when both proteins are transiently expressed in COS cells to investigate which parts of the AChR might interact with the 43 kDa protein. By constructing chimeric subunits, we showed that the cytoplasmic domains of neither the epsilon nor delta subunits are required for 43 kDa protein-induced clustering. Systematic mutational analysis of the long cytoplasmic loops of the alpha and beta subunits showed that most of the loops can be altered without affecting the ability of the AChR to be clustered; in each case, however, one or more sequences could not be tested, because mutation in these regions prevented AChR assembly. Our results suggest either that these regions are involved in clustering or that the 43 kDa protein can interact with multiple, alternative sites on the cytoplasmic surface of the AChR. Our experiments also show that the postulated sites of tyrosine phosphorylation in the beta subunit and of serine phosphorylation in the alpha subunit can be mutated without affecting 43 kDa protein- induced AChR clustering.