The mechanisms by which replicating influenza viruses decrease the expression and function of amiloride-sensitive epithelial sodium channels (ENaCs) have not been elucidated. We show that expression of M2, a transmembrane influenza protein, decreases ENaC membrane levels and amiloride-sensitive currents in both Xenopus oocytes, injected with human -, -, and -ENaCs, and human airway cells (H441 and A549), which express native ENaCs. Deletion of a 10-aa region within the M2 C terminus prevented 70% of this effect. The M2 ENaC down-regulation occurred at normal pH and was prevented by MG-132, a proteasome and lysosome inhibitor. M2 had no effect on Liddle ENaCs, which have decreased affinity for Nedd4-2. H441 and A549 cells transfected with M2 showed higher levels of reactive oxygen species, as shown by the activation of redox-sensitive dyes. Pretreatment with glutathione ester, which increases intracellular reduced thiol concentrations, or protein kinase C (PKC) inhibitors prevented the deleterious effects of M2 on ENaCs. The data suggest that M2 protein increases steady-state concentrations of reactive oxygen intermediates that simulate PKC and decrease ENaCs by enhancing endocytosis and its subsequent destruction by the proteasome. These novel findings suggest a mechanism for the influenza-induced rhinorrhea and life-threatening alveolar edema in humans.—Lazrak, A., Iles, K. E., Liu, G. Noah, D. L., Noah, J. W., Matalon, S. Influenza virus M2 protein inhibits epithelial sodium channels by increasing reactive oxygen species.