We investigated whether adenovirus-mediated transfer of genes encoding for subunits of the Na,K-ATPase increases transepithelial Na⁺ transport in rat fetal distal lung epithelial (FDLE) monolayers and renders them more resistant to hydrogen peroxide injury. FDLE cells, isolated from rat fetuses at a gestational age of 19 to 20 d (22 d = term), were seeded on filters and infected with replication-incompetent human type 5 adenoviruses containing complementary DNAs encoding for rat Na,K-ATPase α₁ or β₁ subunits (ad α₁ and ad β₁, respectively). Once confluent monolayers were formed, the filters were mounted in Ussing chambers and short circuit currents (I_SC) were measured. Increased levels of α₁ or β₁ subunit proteins after infection with ad α₁ and ad β₁, respectively, were confirmed by Western blot analysis. Baseline I_SC increased after transfection with 2 plaque-forming units (pfu) of ad β₁ from 5.1 ± 0.3 to 6.1 ± 0.3 μ A/cm² (mean ± SEM; P < 0.05). Permeabilization of the apical membrane with amphotericin B caused a large increase in I_SC; the ouabain-sensitive component of the amphotericin B–elicited I_SC (ouab_max) was increased from 4.0 ± 0.2 (n = 69) in controls to 4.8 ± 0.2 (n = 15), 5.9 ± 0.3 (n = 53), 6.9 ± 0.4 (n = 25), 7.7 ± 0.9 (n = 16) in monolayers infected with 1, 2, 11, and 22 pfu of ad β₁, respectively; transfection with ad α₁ had no effect on any measured variables. Further, transfection with ad β₁ in comparison to noninfected monolayers resulted in higher baseline and ouab_max I_SC after injury with 500 μ M H₂O₂. We conclude that overexpression of the β₁ subunit of the Na,K-ATPase may help maintain normal levels of vectorial Na⁺ transport across ATII cell monolayers in pathologic conditions.