We used siRNA against the α-ENaC (epithelial Na channel) subunit to investigate ENaC involvement in lung fluid absorption in rats by the impermeable tracer technique during baseline and after β-adrenoceptor stimulation by terbutaline. Terbutaline stimulation of lung fluid absorption increased fluid absorption by 165% in pSi-0-pretreated rat lungs (irrelevant siRNA-generating plasmid). Terbutaline failed to increase lung fluid absorption in rats given the specific α-ENaC siRNA-generating plasmid (pSi-4). pSi-4 pretreatment reduced baseline lung fluid absorption by ∼30%. α-ENaC was undetectable in pSi-4-pretreated lungs, regardless of condition but was normal in pSi-0-pretreated lungs. We carried out a dose-response analysis where rats were given 0–200 μg/kg body wt pSi-4, and α-ENaC mRNA and protein expressions were analyzed. To reach IC₅₀ for α-ENaC mRNA expression, 32 μg/kg body wt pSi-4 was needed, and to reach IC₅₀ for α-ENaC protein expression, 59 μg/kg body wt pSi-4 was needed. We tested for lung tissue specificity and found no changes in β-ENaC expression, at either mRNA or protein level, as well as no changes in α₁-Na-K-ATPase protein expression. We isolated alveolar epithelial type II cells 24 h after in vivo pSi-4 pretreatment. In these cells, α-ENaC mRNA was undetectable, demonstrating that alveolar epithelial ENaC expression was attenuated after intratracheal α-ENaC siRNA-generating plasmid DNA instillation. We tested for organ specificity and found no changes in kidney α- and β-ENaC mRNA and protein expression. Thus we provide conclusive evidence that β-adrenoceptor stimulation of lung fluid absorption is critically ENaC dependent, whereas baseline lung fluid absorption seemed less ENaC dependent.