Mechanical ventilation with high tidal volumes (HV_T) downregulates alveolar Na,K-ATPase function and impairs lung liquid clearance. We hypothesized that overexpression of Na,K-ATPase in the alveolar epithelium could counterbalance these changes and increase clearance in a rat model of mild ventilation-induced lung injury. We used a surfactant-based system to deliver 4 × 10⁹ plaque-forming units of E1a⁻/E3⁻ recombinant adenovirus containing either a rat β₁ Na,K-ATPase subunit cDNA (adβ₁) or no cDNA (adnull) to rat lungs 7 days before ventilation with a V_T of approximately 40 ml/kg (peak airway pressure of less than 35 cm H₂O) for 40 minutes. Lung liquid clearance and Na, K-ATPase activity and protein abundance were increased in HV_T adβ₁-infected lungs as compared with sham and adnull-infected HV_T lungs. These results suggest that Na,K-ATPase subunit gene overexpression in the alveolar epithelium increases Na,K-ATPase function and lung liquid clearance in a model of HV_T. We provide here the first evidence that using a genetic approach improves active Na⁺ transport and thus liquid clearance in the setting of mild ventilation-induced lung injury.