The predicted major intracellular domains of the chick and rat neuronal nicotinic acetylcholine receptor alpha 7 subunits were expressed in E. coli as glutathione-S-transferase fusion proteins. These proteins were then purified to near homogeneity by chromatography on immobilized glutathione. The intracellular domains of the alpha 7 subunit from both species were phosphorylated to high stoichiometry by cAMP-dependent protein kinase, but not by protein kinase C, cGMP-dependent protein kinase, or calcium/calmodulin-dependent protein kinase. Phosphorylation occurred on serine residues only within an identical single tryptic peptide for both proteins. This conserved phosphorylation site was identified as Ser 342 utilizing site-directed mutagenesis. These results demonstrate that the intracellular domain of the alpha 7 subunit is a substrate of PKA, and suggest a role for protein phosphorylation in mediating cellular regulation upon neuronal AChRs containing this subunit.