BACKGROUND: Na+ transport via the amiloride-sensitive epithelial Na+ channel (ENaC) regulates airway surface liquid (ASL) volume and mucus clearance. In cystic fibrosis (CF) airways, increased sodium absorption depletes the ASL resulting in dysfunctional mucocilliary clearance and bacteria colonisation of the lung. Although serine protease such as prostasin is an important regulator of ENaC activity, it is largely inactive at pH seen in the acidic environment of CF airways. Here, we investigate the effect of pH and Cathepsin B (CTSB) activity on the regulation of ENaC. METHODS: The activity and expression of CTSB in polarised primary human bronchial epithelial cultures (HBECs; normal and CF) and ASL lavage were examined using fluorogenic substrates that are specifically cleaved by CTSB (Z-Arg-Arg-MCA, 50 μM) and western blotting respectively. We studied the effects of CTSB on amiloride-sensitive ENaC whole-cell currents (ΔIami) using two voltage clamp and cleavage of ENaC by surface biotinylation in Xenopus oocytes. To demonstrate the physiological relevance of CTSB in polarised HBECs, we used confocal microscopy to measure the ASL height in the presence and absence of a cell-impermeable CTSB inhibitor (CA074, 10 μM). RESULTS: The activity of CTSB on the apical membrane surface and ASL lavage harvested from normal and CF polarised HBECs was significantly greater at pH 6 compared to pH 7.5, 7 or 6.5. Western blotting revealed that CTSB was secreted into the ASL in both normal and CF HBECs. Basal ΔIami in oocytes expressing rat αβγENaC and CTSB exposed to pH 6 or pH 7.3 were not significantly different. However, coexpression of ENaC and CTSB stimulated ΔIami by approximately 6-fold from 681.7 ± 61.4 to 4143.3 ± 417.1 nA (p < 0.0001, n = 6) at pH 7.3. In the presence of hCTSB, trypsin, but not chymotrypsin, further stimulated the ΔIami. Importantly, western blotting indicated a reduction in the molecular weight of surface-expressed γENaC in oocytes expressing ENaC and CTSB. In normal and CF HBECs, exposure to pH 6 Ringer solution significantly affect ASL regulation and reduced height from 13.4 ± 1.1 to 11.4 ± 0.9 μm (p < 0.05, n = 5) and 12.1 ± 0.8 to 7.5 ± 0.5 μm (p < 0.05, n = 5), respectively. CA074 significantly increased the ASL height in normal (11.4 ± 0.9 to 15.2 ± 1.5 μm, p < 0.05, n = 7) and CF (7.5 ± 0.5 to 11.5 ± 0.6 μm, p < 0.01, n = 7) HBECs at pH 6 but not pH 7.5. CONCLUSIONS: Taken together, these results indicate that CTSB, is secreted into the ASL, is more active at acidic pH and can regulate ASL height through the cleavage of γENaC. This is the first study to implicate CTSB in the pathphysiology of CF, suggesting that inhibitors of CTSB could potentially be used for the treatment of the disease.