Glutathione (GSH) plays vital roles in antioxidant defense mechanisms. Premature infants have low levels of GSH that may predispose them to oxidative injury. GGCS, which exists as a dimer of heavy (Hc)and light chain subunits, is the rate-limiting enzyme in GSH synthesis; the Hc subunit contains the active site. To determine whether enhanced GSH synthetic capacity would augment antioxidant defense functions, we used liposome-mediated transfer of the cDNA for rat GGCS-Hc subunit to transfer Chinese hamster ovary (CHO) cells and increased GGCS activities from 0.50±0.09 (mean±SD) mU/mg pro to 10.63±2.96 (n=4-6). CHO cell lines with stably enhanced GGCS activities (GGCS-h) were developed by transfection with vectors containing the GGCS-Hc cDNA and hygromycin/neomycin resistance genes. Southern analyses of the GGCS-h cells indicate the presence of the transfected transcript, and Northern analyses indicate transcription of the transfected GGCS gene. The effects of enhanced GGCS activities in these cells on resistance to oxidant-mediated cytotoxicity was assessed by exposure of cells to t-BuOOH or DQ. After exposure to 0.5 or 1 mM t-BuOOH for 12 h, native CHO cells had released more cellular LDH (62.1±0.7% and 76.8±1.3%) than had GGCS-h cells (21.6±2.7% and 37.6±1.6%) (n=4). After 26 h of exposure to DQ, survival, as assessed by a tetrazolium reduction assay was lower in the parent CHO cells than in GGCS-h cells at DQ concentrations of 1, 2 or 4 mM (21.2, 6.5, 0.6% vs. 27.8, 19.4, 13.8%, respectively), but not at 0.1 or 0.5 mM (62.2 and 28.4% vs 40.2 and 30.1%). Studies of cellular GSH contents demonstrated depletion of GSH concomitant with the onset of cytotoxicity. Conclusion: Our results indicate that increased GSH synthetic capacity in mammalian cells produced by transfection with GGCS heavy chain cDNA offers significant protection against the cytotoxic actions of oxidants added exogenously (t-BuOOH), or generated endogenously in response to redox cycling metabolism of diquat. The paradoxical response of the GGCS-h cells to DQ may relate to the intracellular compartmentalization of critical targets and antioxidant systems. (We acknowledge Wyeth Neonatology Research Grants Program and GM44263 and HD27823).