Oxidative stress is a likely contributor to the pathogenesis of cystic fibrosis (CF) lung disease. However, hydrogen peroxide (H₂O₂), a physiological oxidant, is not elevated in CF exhalates. H₂O₂ may be neutralized by antioxidants in CF airway secretions. The H₂O₂-detoxifying capacity of CF airway secretions, obtained via sputum induction, was studied in an in vitro H₂O₂ cytotoxicity model. 16HBE14o- cells were exposed to H₂O₂ in culture medium containing either 0 or 10% fetal bovine serum (FBS) or 10% CF sputum supernatant (extracted without use of dithiothreitol). The efficiency of H₂O₂ neutralization was estimated by measuring intracellular oxidant levels (dihydrorhodamine 123) after 2 hours and cell viability (propidium iodide) after 24 hours of H₂O₂ exposure. Further, the presence of reduced thiols (DTNB assay) and reduced glutathione (recycling assay) in CF sputum samples was evaluated. CF sputum extracts completely prevented intracellular oxidant accumulation seen in cells incubated with H₂O₂ in both control media (i.e. 0 or 10% FBS). Further, CF sputum abolished cell death in 16HBE14o- cells exposed to up to 1 mM H₂O₂. In contrast, there was a 100% cytotoxicity in cells exposed to 600 µM H₂O₂ in both control media. The H₂O₂-detoxifying potential of CF sputum was sustained after catalase and heme peroxidases were inactivated by sodium azide, which does not affect glutathione peroxidase. In addition, reduced protein thiols were found in abundance in CF sputum. In conclusion, CF sputum is capable to neutralize H₂O₂ and abundant reduced thiols and/or glutathione peroxidase are fully sufficient to detoxify H₂O₂.