To explore interactions between ENaC (epithelial Na channel) and Nedd4-2 (neural precursor expressed, developmentally down-regulated protein 4-2) at the conversion of the rat lung from fluid secretion to absorption at birth, we used small interfering RNA (siRNA) against ENaC and Nedd4-2. siRNA-generating plasmid DNA (pDNA) was administered via trans-thoracic intrapulmonary (ttip) injection 24 h before ENaC and Nedd4-2 expression, extravascular lung water, and mortality were measured. ENaC mRNA and protein were specifically reduced by ~80% after pSi-4 injection. Nedd4-2 mRNA and protein were reduced by ~50% after pSi-N1 injection. Interestingly, ENaC and ENaC mRNA and protein expression were increased after Nedd4-2 silencing. Extravascular lung water was significantly increased after ENaC silencing and reduced after Nedd4-2 silencing. ENaC silencing resulted in a four-fold increase in newborn mortality, while silencing Nedd4-2 did not affect mortality. We also isolated distal lung epithelial (DLE) cells after in vivo ENaC or Nedd4-2 silencing and measured ENaC or Nedd4-2 expression in freshly isolated DLE cells. In these DLE cells there were attenuated ENaC or Nedd4-2 mRNA and protein, thus demonstrating that ENaC and Nedd4-2 silencing occurred in alveolar epithelial cells after ttip injection. We also looked for pDNA by PCR to determine pDNA presence in the lungs and found strong evidence for pDNA presence in both lungs. Thus, we provide evidence that ENaC and Nedd4-2 are involved in the transition from lung fluid secretion to fluid absorption near term and at birth.