Recent studies suggest that the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein modulates epithelial reduced glutathione (GSH) transport and when defective creates an antioxidant imbalance. To test whether the CFTR protein modulates lung antioxidant defenses in vivo, epithelial lining fluid (ELF) and lung tissue from CFTR knockout (CFTR-KO) and wild-type (WT) mice were compared for GSH content and the activities of glutathione reductase, glutathione peroxidase, and gamma-glutamyltransferase. In the CFTR-KO mice, the ELF concentration of GSH was decreased (51%) compared with that in WT mice. The concentration of GSH in the lung tissue of CFTR-KO mice, however, was not significantly different from that in WT mice. The activities of glutathione reductase and glutathione peroxidase in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice (48 and 28%, respectively). Tissue lipid and DNA oxidation were evaluated by measurement of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine, respectively. The levels of thiobarbituric acid-reactive substances and 8-hydroxy-2'-deoxyguanosine in the lung tissue of CFTR-KO mice were significantly increased compared with those in WT mice. These data support our hypothesis that a mutation in the CFTR gene can affect the antioxidant defenses in the lung and may contribute to the exaggerated inflammatory response observed in CF.