Neonatal organisms have exaggerated NF-B activation following oxidant and inflammatory insults, but the reason for this and the down-stream effects are unclear. We hypothesized that specific phosphorylation patterns of IB could account for differences in NF-B activation in hyperoxia-exposed fetal and adult lung fibroblasts. Following hyperoxic exposure (O₂ > 95%), nuclear NF-B binding increased in fetal but not adult lung fibroblasts. Unique to fetal cells, phosphorylation of IB on tyrosine 42, rather than serine 32/36 as seen in TNF- exposed cells, preceded NF-B nuclear translocation. In fetal cells stably transfected with a NF-B-driven luciferase reporter, hyperoxia significantly suppressed reporter activity, in contrast to increased reporter activity after TNF- incubation. Targeted gene profiling analysis showed that hyperoxia resulted in decreased expression of multiple genes, including pro-apoptotic factors. Transfection with a dominant negative IB (Y42F), which cannot be phosphorylated on tyrosine 42, resulted in up-regulation of multiple pro-apoptotic genes. In corroboration, caspase-3 activity and DNA laddering was specifically increased in fetal lung fibroblasts expressing Y42F following exposure to hyperoxia. These data demonstrate a unique pathway of NF-B activation in fetal lung fibroblasts following exposure to hyperoxia, whereby these cells are protected against apoptosis. Activation of this pathway in fetal cells may prevent the normal pattern of fibroblast apoptosis necessary for normal lung development, resulting in aberrant lung morphology in vivo.