Pulmonary edema is cleared via active Na⁺ transport by alveolar epithelial Na⁺/K⁺-ATPases and Na⁺ channels. Rats exposed to acute hyperoxia have a high mortality rate, decreased Na⁺/K⁺-ATPase function, and decreased alveolar fluid clearance (AFC). We hypothesized that Na⁺/K⁺-ATPase subunit gene overexpression could improve AFC in rats exposed to hyperoxia. We delivered 4 × 10⁹ PFU of recombinant adenoviruses containing rat α₁ and β₁ Na⁺/K⁺-ATPase subunit cDNAs (adα₁ and adβ₁, respectively) to rat lungs 7 days prior to exposure to 100% O₂ for 64 hr. As compared with controls and ad α₁, AFC in the adβ₁ rats was increased by >300%. Permeability for large solutes was less in the ad β₁ than in the other hyperoxia groups. Glutathione oxidation, but not superoxide dismutase activity, was increased only in the adβ₁ group. Survival through 14 days of hyperoxia was 100% in the adβ₁ group but was not different from hyperoxic controls in animals given adα₁. Our data show that overexpression of a β₁ Na⁺/K⁺-ATPase subunit augments AFC and improves survival in this model of acute lung injury via antioxidant-independent mechanisms. Conceivably, restoration of AFC via gene transfer of Na⁺/K⁺-ATPase subunit genes may prove useful for the treatment of acute lung injury and pulmonary edema.