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, H₂O₂ levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air–liquid interface were estimated, and H₂O₂-mediated changes in CFTR activity were examined. In Ussing chambers, H₂O₂-induced anion currents were sensitive to the CFTR inhibitors CFTR_inh172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Responses to greater than 500 μM H₂O₂ were transient. Cyclooxygenase inhibitors blocked the H₂O₂ response, as did EP1 and EP4 receptor antagonists. A multidrug-resistant protein (MRP) inhibitor and short hairpin RNA directed against MRP4 blocked H₂O₂ responses. EP1 and EP4 agonists mimicked H₂O₂ in both control and MRP4 knockdown cells. Thus, H₂O₂ activates the synthesis, export, and binding of prostanoids via EP4 and, interestingly, EP1 receptors in normal, differentiated human airway epithelial cells to activate cyclic adenosine monophosphate pathways that in turn activate CFTR channels in the apical membrane.