The alveolar macrophage is an important source of interleukin (IL)-8 during pulmonary injury. The IL-8 gene promoter sequence contains nuclear factor (NF) B, NF-IL6 and AP-1 binding sequences. These sites may have differing regulatory roles in hyperoxia-exposed macrophages than in those stimulated by bacterial lipopolysaccharide (LPS). U937 and THP-1 macrophage-like cells were exposed to 95% air/5% CO₂ or 95% O₂/5% CO₂, with or without LPS 1.0 µg/mL, and transfected with an IL-8 promoter-reporter containing NFB, NF-IL6 or AP-1 mutations. Hyperoxia and LPS caused additive increases in IL-8 production by U937 cells, while THP-1 cells responded only to LPS. An NFB mutation ablated baseline and O₂- and LPS-stimulated reporter activity in both cell lines, while NF-IL6 mutation had little effect. An AP-1 mutation had an intermediate effect. LPS, but not hyperoxia, stimulated nuclear translocation of NFB in both cell lines. Pharmacological blockade of NFB nuclear translocation ablated LPS-, but not hyperoxia-, stimulated IL-8 production. Although an intact promoter NFB site is crucial to macrophage IL-8 production, only LPS-stimulated production appears to require additional nuclear translocation of NFB.