Rationale: Na⁺ absorption and Cl^- secretion are in equilibrium to maintain an appropriate airway surface fluid volume and ensure appropriate mucociliary clearance. In cystic fibrosis, this equilibrium is disrupted by mutations in the CFTR gene resulting in the absence of functional CFTR protein which in turn results in deficient cAMP-dependent Cl^- secretion and predominant Na⁺ absorption. It has been suggested that downregulation of the epithelial sodium channel, ENaC, might help to restore airway hydration and reverse the airway phenotype in cystic fibrosis patients. Objectives and Methods: Here we have used a siRNA approach to analyse the possibility to downregulate ENaC function in bronchial epithelia and examine the resulting effects on fluid transport. siRNA sequences complementary to each of the three ENaC subunits have been used to establish whether single subunit downregulation is enough to reduce Na⁺ absorption. Transfection was performed by exposure to siRNA for 24 h at the time of cell seeding on permeable support. Measurements and Main Results: By using primary human bronchial epithelial cells we demonstrate that i) siRNA sequences complementary to ENaC subunits are able to reduce ENaC transcripts and Na⁺ channel activity by 50-70%, 2) that transepithelial fluid absorption decreases, and 3) that these functional effects last at least 8 days. Conclusions: A decrease in ENaC mRNA results in a significant reduction of ENaC protein function and fluid absorption through the bronchial epithelium, indicating that an RNA interference approach may improve the airway hydration status in cystic fibrosis patients.