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 cystic fibrosis transmembrane conductance regulator (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 down-regulation of the epithelial sodium channel, ENaC, might help to restore airway hydration and reverse the airway phenotype in patients with cystic fibrosis. We used an siRNA approach to analyze the possibility of down-regulating 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 down-regulation is enough to reduce Na(+) absorption. Transfection was performed by exposure to siRNA for 24 hours at the time of cell seeding on permeable support. By using primary human bronchial epithelial cells we demonstrate that (1) siRNA sequences complementary to ENaC subunits are able to reduce ENaC transcripts and Na(+) channel activity by 50 to 70%, (2) transepithelial fluid absorption decreases, and (3) these functional effects last at least 8 days. 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 patients with cystic fibrosis.