To determine whether α4 subunits of α4β2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of α4 (α4336−597) were incubated with ATP and either PKA or PKC. Both α4 and α4336−597 were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor α4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the α4336−597 fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that α4 and α4336−597 were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser368 was a substrate for both kinases, a peptide corresponding to amino acids 356–371 was synthesized (α4356−371) and incubated with ATP and the kinases. The phosphorylation of α4356−371 by both PKA and PKC was saturable with Kms of 15.3 ± 3.3 µm and 160.8 ± 26.8 µm, respectively, suggesting that Ser368 was a better substrate for PKA than PKC.