We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na⁺ reabsorption during water conservation vs. maintenance of Na⁺ balance. We find that vasopressin significantly increases ENaC activity within 2–3 min by increasing open probability (P_o). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18–24 h) and pretreatment of isolated CD with vasopressin (∼30 min) resulted in a similar increase in P_o. In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P_o, increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na⁺ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na⁺ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.