This study determined whether the α4 subunit of human α4β2 neuronal nicotinic receptors is phosphorylated in situ by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). To accomplish this, human cloned epithelial cells stably transfected with the human α4β2 nicotinic receptor (SH-EP1-hα4β2) were incubated with 3 2 P-orthophosphate to label endogenous ATP stores, and the phosphorylation of α4 subunits was determined in the absence or presence of PKA or PKC activation. Autoradiographs and immunoblots indicated that α4 subunits immunoprecipitated from a membrane preparation of SH-EP1-hα4β2 cells exhibited a single 3 2 P-labeled band corresponding to the α4 subunit protein; no signals were associated with untransfected SH-EP1 cells. The α4 subunits from SH-EP1-hα4β2 cells incubated in the absence of the activators exhibited a basal level of phosphorylation that was decreased in the presence of the PKA inhibitor H-89 (5 μM), but unaltered in the presence of the PKC inhibitor Ro-31-8220 (0.1 μM). Activation of PKA by forskolin (10 μM), dibutyryl-cAMP (1 mM), or Sp-8-Br-cAMP (1 mM) enhanced phosphorylation nearly threefold; the inactive isomer, Rp-8-Br-cAMP (1 mM) had no effect. In addition, the forskolin effect was totally blocked by the PKA inhibitor H-89 (5 μM). Activation of PKC by the phorbol esters PDBu (200 nM) or PMA (200 nM) increased α4 subunit phosphorylation approximately twofold, and the PDBu effect was blocked by the selective PKC inhibitor Ro-31-8220 (0.1 μM). These findings indicate that the α4 subunit of human α4β2 nicotinic receptors is phosphorylated in situ by PKA and PKC.