Mucus clearance is an important aspect of the mammalian lung’s innate defense system. During normal mucus clearance, inhaled pathogens become trapped in the mucus layer and are expelled before they can colonize the airways. The mucus layer is kept away from underlying epithelia by the presence of a ~7 μm 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). 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. Importantly, these reporter molecules are contained within the ASL and are lost when cultures are mounted in Ussing chambers or following other experimental protocols that require flooding of the mucosal surface. The study of normal and CF airway epithelia under thin film conditions is essential to understanding the regulation of CFTR and ENaC and studies of airway epithelia under flooded conditions may produce aberrant results. In contrast, thin film studies correlate well with the increased cleavage pattern seen with ENaC in CF airways biochemically. 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 serves to prevent excessive volume absorption when ASL volume is at optimum levels. Removal or dilution of SPLUNC1 leads to increased ASL absorption and temporary ENaC hyperactivity in normal airway cultures – until SPLUNC1 level rise again. For reasons that are currently under investigation, SPLUNC1 is present in CF ASL but is unable to regulate ENaC in CF airway cultures. To better understand defective SPLUNC1 regulation in CF, we identified the ENaC inhibitory domain of SPLUNC1 and further proteomic analysis suggested that this domain may be functionally deficient in CF airways in vivo. In addition to being a Cl- channel, CFTR is also permeable to bicarbonate, and CF airways are hypothesized to 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 airway epithelia. In this talk, we will discuss how the interaction between soluble reporter molecules such as SPLUNC1 and ENaC are affected by pH. Furthermore, many extracellular proteases are pH-sensitive and their impact on ENaC and ASL volume will also be discussed.