The participation of protein kinases in phosphorylation of nicotinic acetylcholine receptor (nAChR) in electric organ and muscle has been precisely investigated in vitro and in vivo whereas phosphorylation of neuronal nAChR is not yet fully characterized. Here, we first report the in vitro phosphorylation of brain nAChR. nAChR purified from rat brains was phosphorylated in vitro by cAMP-dependent protein kinase (PKA), immunoprecipitated with monoclonal antibody against the receptor, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. PKA specifically phosphorylated nAChR on the alpha 4 subunits, and H8, an inhibitor of PKA, inhibited completely the phosphorylation. Under the conditions used, a maximal stoichiometry of the phosphorylation by PKA was near to 1 mol of phosphate/mol of the alpha 4 subunits. The 32P-labeled subunits were digested with S. aureas V8 protease followed by SDS-PAGE autoradiography and the resultant phosphopeptide maps revealed three distinct phosphopeptide bands, one major band and two minor bands. Phosphoamino acid analysis of the 32P-labeled alpha 4 subunits showed that serine residues were exclusively phosphorylated. Based on these results, participation of PKA in the regulation of neuronal nAChR is discussed.