In normal lungs, inhaled pathogens become trapped in the mucus layer and are expelled before they can colonize the airways. The mucus layer is kept hydrated by the presence of a ~7 micron periciliary liquid layer (PCL), which acts as a lubricant for the mucus. Together, these layers make up the airway surface liquid (ASL). The rate of mucociliary clearance is strongly influenced by the hydration state of the ASL. ASL volume hydration is largely maintained by Cl- secretion through CFTR, with Na+ and H2O following by osmosis. However, as ASL moves up the respiratory tract, due to a reduction in surface area as airways converge, excess ASL is absorbed when needed via isotonic Na+-led ASL absorption, which is mediated by the epithelial Na+ channel (ENaC). In addition to being a Cl- channel, CFTR is also permeable to bicarbonate, and CF airways have a reduced ASL pH due to a lack of bicarbonate secretion through CFTR. This reduction in pH may be minimized by the backflow of bicarbonate through the paracellular pathway. Nonetheless, differences in pH in excess of 0.5 pH unit difference have been detected between normal and CF ASL. Our experimental data indicate that ASL volume and ion transport regulation are in dynamic equilibrium, and that either a reduction in CFTR-mediated Cl- secretion or an increase in ENaC-mediated Na+ absorption will lead to ASL/PCL depletion and mucus dehydration. ENaC is highly sensitive to both membrane attached and soluble extracellular proteases which can cleave and activate ENaC. In addition, soluble volume sensing reporter molecules are present in the ASL and can bind to ENaC when ASL volume is low, removing ENaC from the plasma membrane, thus favouring secretion. These reporter molecules act as feedback sensors to modulate ASL volume. We have recently identified the short palate lung nasal epithelial clone 1 (SPLUNC1) as one such ASL volume-sensing molecule. SPLUNC1 acts as a potent negative regulator of ENaC that protects ENaC from proteolysis and serves to prevent excessive ASL volume absorption. Knockdown of SPLUNC1 leads to CF-like increased ASL absorption and ENaC hyperactivity in normal airway cultures. SPLUNC1 is present in CF ASL but is unable to regulate ENaC in these cultures. To better understand defective SPLUNC1 regulation in CF, we identified the ENaC inhibitory domain of SPLUNC1, which coincides with SPLUNC1’s N-terminus. Interestingly, while SPLUNC1 fails to regulate ENaC under the acidic conditions seen in CF airways, a peptide corresponding to SPLUNC1’s ENaC inhibitory domain is pH-independent. Further analysis of SPLUNC1’s crystal structure revealed the presence of pH-sensitive salt bridges which confers pH-sensitivity on SPLUNC1. We conclude that the acidic environment seen in CF airways due to the lack of bicarbonate secretion through CFTR prevents SPLUNC1 from regulation ENaC, leading to Na+ hyperabsorption and ASL volume depletion. Our data suggest that therapies for the treatment of CF lung disease should be directed at normalizing CF ASL pH.