The nicotinic acetylcholine receptor (AChR) from the electric organ of T. californica is highly phosphorylated on tyrosine residues in vivo. In contrast, tyrosine phosphorylation of the AChR in rat myotube cultures is barely detectable. To determine whether this low level of tyrosine phosphorylation of the AChR in muscle cell cultures is due to a lack of neuronal innervation, we examined tyrosine phosphorylation of the AChR in rat diaphragm in vivo. Immunofluorescent double labeling of cryostat sections of rat diaphragm using antibodies specific for phosphotyrosine or the AChR showed a direct colocalization of phosphotyrosine with the AChR at the neuromuscular junction. Using anti-phosphotyrosine antibodies, immunoblots of AChR partially purified from rat diaphragm demonstrated that the rat AChR contains high levels of phosphotyrosine. Denervation of rat diaphragm induced a time-dependent decrease in tyrosine phosphorylation of the AChR, as measured by immunocytochemical and immunoblot techniques. Tyrosine phosphorylation of the AChR occurred late in the development of the neuromuscular junction, between postnatal days 7 and 14. These studies suggest that muscle innervation regulates tyrosine phosphorylation of the AChR and that tyrosine phosphorylation may play an important role in the developmental regulation of the AChR.