Voltage-sensitive brain Na+ channels are regulated by cAMP-dependent protein kinase (cA-PK) and protein kinase C. Using synthetic peptides and protein microsequencing, we have determined that the alpha subunit of rat brain Na+ channel is selectively phosphorylated by cA-PK in vitro and in intact cells on 4 serine residues in the intracellular loop connecting homologous domains I and II. Ser-623 was most rapidly and extensively phosphorylated in vitro, whereas Ser-573, Ser-610, and Ser-687 were phosphorylated to lesser extents. In contrast, serine 687 was most extensively phosphorylated in mammalian cells transfected with the alpha subunit of type IIA Na+ channel in response to an increase in intracellular cAMP. Purified protein phosphatases dephosphorylated these sites selectively. Calcineurin rapidly and extensively dephosphorylated Ser-623 and also dephosphorylated Ser-573, Ser-610, and Ser-687 to lesser extents. Phosphatase 2A selectively dephosphorylated Ser-610. Together these results indicate that modulation of neuronal Na+ channel activity and therefore neuronal excitability by cAMP-dependent phosphorylation results from selective phosphorylation and dephosphorylation of four sites in the intracellular loop connecting homologous domains I and II of the alpha subunit.