The activity of the epithelial sodium channel (ENaC) in the distal nephron is regulated by an antidiuretic hormone, aldosterone, and insulin, but the molecular mechanisms that mediate these hormonal effects are mostly unknown. We have investigated whether aldosterone, insulin, or activation of protein kinases has an effect on the phosphorylation of the channel. Experiments were performed in an epithelial cell line generated by stable cotransfection of the three subunits (α, β, and γ) of ENaC. We found that β and γ, but not the α subunit, are phosphorylated in the basal state. Aldosterone, insulin, and protein kinases A and C increased phosphorylation of the β and γ subunits in their carboxyl termini, but none of these agents induced de novo phosphorylation of α subunits. Serines and threonines but not tyrosines were found to be phosphorylated. The results suggest that aldosterone, insulin, and protein kinases A and C modulate the activity of ENaC by phosphorylation of the carboxyl termini of the β and γ subunits.