Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H 2 O 2 levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air-liquid interface were estimated, and H 2 O 2 -mediated changes in CFTR activity were examined. In Ussing chambers, H 2 O 2 -induced anion currents were sensitive to the CFTR inhibitors CFTR inh 172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Effective airway clearance relies on the regulation of ciliary beat frequency (CBF), adequate airway surface liquid (ASL) volume, and correct mucus properties. Mucus hydration and thus the viscosity of mucus depend, in part, on the movement of water through the airway epithelium to the mucosal surface via the coordinated activities of ion channels. An important player in this regulation is the cystic fibrosis transmembrane conductance regulator (CFTR) that passes chloride (and other anions) while regulating the epithelial Na 1 channel to balance osmolarity (1). CFTR and CBF are both regulated through increases in cyclic adenosine monophosphate (cAMP) and protein kinase A activity (2-4). Thus, mediators that stimulate adenylyl cyclase are expected to result in improved airway clearance by increasing chloride secretion and CBF.Airway epithelia actively synthesize H 2 O 2 via the nicotinamide adenine dinucleotide phosphate-reduced (NADPH) dual oxidases Duox1 and Duox2, as reviewed by Fischer (5), presumably for the use of the lactoperoxidase host defense against infection (6-9). Duox1 and Duox2 are regulated by cytokines (10-12) and bacterial products (10,13,14) to increase H 2 O 2 synthesis to high levels (10). Stimulation with interferon-g increased H 2 O 2 synthesis levels 30-fold when measured in air-liquid interface (ALI) cultures of normal human airway epithelial cells (10), leading to 10 mM H 2 O 2 in washes used for assays. The experiments presented here address ASL H 2 O 2 and show that it is present at high concentrations.In addition to its role in stimulating lactoperoxidase host defense, H 2 O 2 may also regulate the innate defense related to mucociliary clearance. In the bronchial submucosal adenocarcinoma cell line Calu-3, CFTR activity is increased by exposure of the airway epithelium to exogenous H 2 O 2 (15, 16). In cell lines, the effect of H 2 O 2 on CFTR is reported to occur through increases in transmembrane adenylyl cyclase activity and through the activation of two K 1 channels (15), most likely KCNQ1 and KCNN4, which are located in the basolateral membrane. These channels are believed to provide a driving force for apical Cl 2 release (17, 18). Yet the mechanisms by which H 2 O 2 leads to the activation of the cAMP pathway are not understood. Although both Calu-3 cells (19) and bovine tracheal epithelia (20) have been shown to respond to isop...