NEW FINDINGS: What is the central question of this study? What is the precise subcellular localization of the epithelial sodium channel (ENaC) in human airway epithelium? What is the main finding and its importance? ENaC protein has an unexpected localization in the peripheral region of the apical membrane of bronchial epithelial cells, very close to tight junctions. This may be important for the mechanism of Na+ absorption ABSTRACT: The epithelial sodium channel (ENaC) has a key role in absorbing fluid across the human airway epithelium. Altered activity of ENaC may perturb the process of mucociliary clearance, thus impairing the innate defence mechanisms against microbial agents. The proteins forming ENaC are present on the apical membrane of the epithelium. However, their precise localization is unknown. In the present study, we used two antibodies recognizing the α and β ENaC subunits. Both antibodies revealed a restricted localization of ENaC in the peripheral region of the apical membrane of cultured bronchial epithelial cells, close to but not overlapping with tight junctions. In contrast, the cystic fibrosis transmembrane conductance regulator chloride channel was more diffusely expressed on the whole apical membrane. Modulation of ENaC activity by aprotinin or elastase resulted in a decrease or increase in the peripheral localization, respectively. Our results suggest that sodium absorption is mainly occurring close to tight junctions where this cation may be rapidly expelled by the Na+ /K+ pump present in lateral membranes. This arrangement of channels and pumps may limit Na+ build-up in other regions of the cells.