The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine residues both in vitro and in vivo. This phosphorylation appears to regulate the rate of receptor desensitization and is associated with AChR clustering induced by the neuronal extracellular matrix protein agrin. To identify the protein tyrosine kinase(s) that phosphorylates the nicotinic receptor, we have used molecular cloning techniques to identify and characterize two protein tyrosine kinases that are highly expressed in Torpedo electric organ, a tissue enriched in synaptic components including the AChR. One of the kinases was identified as the Torpedo homolog of neuronal fyn, whereas the other was a novel kinase we have named fyk due to its homology to both fyn and yes protein tyrosine kinases. Using antibodies to fyn and fyk, Fyn was shown to be a 55-kDa protein phosphorylated on tyrosine residues, whereas Fyk was a 56-kDa/53-kDa doublet phosphorylated on serine and tyrosine residues. At the mRNA and/or protein level, fyn and fyk were present in Torpedo electric organ, skeletal muscle, and brain. Both kinases were detected in the membrane fractions enriched in the AChR, with Fyn and Fyk representing 36 and 8%, respectively, of the protein tyrosine kinase activity in these postsynaptic membranes. In addition, Fyn and Fyk were shown by coimmunoprecipitation to be specifically associated with the AChR. Furthermore, the AChR was phosphorylated in Fyn and Fyk immunoprecipitates. These results indicate that Fyn and Fyk are involved in the regulation of postsynaptic membrane function and suggest that these protein tyrosine kinases may phosphorylate the AChR.