To test the genetic capacity of the perinatal lung to respond to O₂ shifts that coincide with the first respiratory movements, rat fetal alveolar type II (fATII) epithelial cells were cultured at fetal distal lung P O 2 (23 Torr) and then exposed to postnatal (23 → 76 Torr; mild hyperoxic shift), moderate (23 → 152 Torr; moderate hyperoxic shift), or severe (23 → 722 Torr; severe hyperoxic shift) oxygenation. Nuclear abundance and consensus binding characteristics of hypoxia-inducible factor (HIF)-1α and nuclear factor (NF)-κB (Rel A/p65) plus glutathione biosynthetic capacity were determined. Maximal HIF-1α activation at 23 Torr was sustained over the postnatal shift in (Δ) P O 2 and was elevated in vivo throughout late gestation. NF-κB was activated by the acute postnatal Δ P O 2 in fATII cells, becoming maximal with moderate and severe oxygenation in vitro and within 6 h of birth in vivo, declining thereafter. fATII cell and whole lung glutathione and GSH-to-GSSG ratio increased fourfold with a postnatal Δ P O 2 and were matched by threefold activity increases in γ-glutamylcysteine synthetase and glutathione synthase. GSH concentration depletion byl-buthionine-(S,R)-sulfoximine abrogated both HIF-1α and NF-κB activation, with HIF-1α showing a heightened sensitivity to GSH concentration. We conclude that O₂-linked genetic regulation in perinatal lung epithelium is responsive to developmental changes in glutathione biosynthetic capacity.