The O2 and redox-sensitive transcription factors hypoxia inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) are differentially regulated in the alveolar epithelium over fetal to neonatal oxygen tensions. We have used fetal alveolar type II epithelial cells to monitor their regulation in association with redox responsiveness to antioxidant pretreatment in vitro. N-Acetyl-L-cysteine, a glutathione (GSH) precursor and a potent scavenger of reactive oxygen species, induced HIF-1α and ameliorated NF-κB nuclear abundance and DNA binding activity, respectively, in a dose-dependent manner. Analysis of variations in glutathione homeostasis at ascending ΔPO2 regimen with N-acetyl-(L)-cysteine reveals increased GSH at the expense of the oxidized fore of glutathione (GSSG), thereby shifting GSH/GSSG into reduction equilibrium. Pyrrolidine dithiocarbamate (PDTC), which exerts both antioxidant and pro-oxidant effects, provoked a substantial increase in HIF-1α nuclear abundance, with no apparent effect on its activation. PDTC reduced NF-κB nuclear abundance and its inhibitory effects on binding acridly are dose-dependent. Assessment of glutathione homeostasis with PDTC shows increasing levels of GSSG at the expense of GSH, lowering GSH/GSSG in favor of an oxidative equilibria. Our results indicate the hypoxic activation of HIF-1α and the hyperoxic induction of NF-κB in the fetal epithelium is redox-sensitive and, thus, tightly regulated by the GSH/GSSG equilibrium. This highlights glutathione as a key regulatory component for determining genetic responsiveness to oxidant/antioxidant imbalance in nodal lung development and pathophysiological conditions.