Tyrosine phosphorylation of the beta subunit of the acetylcholine receptor (AChR) has been postulated to play a role in AChR clustering during development of the neuromuscular junction. We have investigated the mechanism of this phosphorylation in mammalian C2 myotubes and report that the tyrosine kinase Src binds and phosphorylates glutathione S-transferase fusion proteins containing the N-terminal half of the cytoplasmic loop of the beta subunit. No binding occurs to the related kinases Fyn or Yes or to the corresponding regions from the gamma and delta subunits. Furthermore, AChRs affinity-isolated from C2 myotubes using alpha-bungarotoxin-Sepharose were specifically associated with Src and Fyn and had tyrosine-phosphorylated beta subunits. We suggest that AChRs are initially phosphorylated by Src and subsequently bind Fyn in a phosphotyrosine-dependent manner. These interactions are likely to play an important role in construction of the specialized postsynaptic membrane during synaptogenesis.