Increased generation of reactive oxygen species (ROS) and low levels of antioxidants may be a major cause of morbidity in premature infants exposed to high concentrations of oxygen. Glutathione (GSH)-dependent antioxidant systems protect against ROS both in the cytoplasm and the mitochondria, and regenerating GSH from glutathione disulfide (GSSG) by the flavoenzyme glutathione reductase (GR) is essential for the optimal function of this system. Previously, we described construction of a vector for human GR cDNA that contains a functional synthetic mitochondrial targeting signal (LGR) and used liposomal gene transfer to increase expression in Chinese hamster ovary(CHO) cells. CHO cells stably transformed with LGR showed greater resistance to t-butyl hydroperoxide (t-BuOOH) induced oxidative stresses. The objectives of the present studies were to determine whether adenovirus-mediated gene transfer could increase GR activities in H441 (lung Clara cell line) cells, and whether such cells would be more resistant to t-BuOOH-induced oxidative stresses. Cells transfected with LGR showed increased cellular GR activities and were protected from t-BuOOH cytotoxicity, as indicated by lower LDH release, than were observed in cells transfected with an irrelevant gene(DOS=superoxide dismutase in the antisense orientation, or a1AT=alpha-1 antitrypsin) or in cells not infected with virus (CON). The total cellular GR activities were 10-fold higher in the LGR-H441 cells (*LGR 458±13; a1AT 41.9±3.5; CON 41.2±3.8 mU/mg pro). Cytosolic GR activities were 5.6-fold higher (*LGR 377.15±2.9; a1AT 56.5±0.3; CON 57.9±2.2), while mitochondrial GR activities were enhanced 10-fold (*GR 230.9±67;a1AT 20.8±6.5; CON 23±0.9). Enhanced GR activities were associated with lower release of LDH in response to t-BuOOH(*LGR 6.6±1.7; DOS 16±1.8; CON 16.6±O.7% LDH release). In addition, cells transfected with LGR retained higher GSH/GSSG ratios (*LGR 66±0.4; DOS 47±1; CON 52.6±2.3) and showed less cellular GSSG efflux (*LGR 0.05±0.01; DOS 0.08±0.01; CON 0.07±0.01 nmol/mg pro) than did DOS or CON cells (*P<0.05, different than other groups). In conclusion, adenovirus-mediated gene transfer of LGR protected H441 cells from oxidant stresses.