The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with (35)S or (32)P. When immune complexes were resolved on SDS-PAGE, the alpha-subunit migrated at 85 and 65 kDa, the beta-subunit at 115 and 100 kDa, and the gamma-subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The alpha-subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of alpha is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of alpha-, beta-, and gamma-subunits approximately twofold. Exposure to 1 microM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport.