Hypoxia and inflammation are coincidental events in a diverse range of disease states including tumor growth, ischemia, and chronic inflammation. Hypoxia contributes to the development of inflammation, at least in part through the activation and/or potentiation of NF-κB, a master regulator of genes involved in innate immunity, inflammation, and apoptosis. NF-κB can be activated through two distinct signaling pathways termed the canonical and noncanonical pathways, respectively. The canonical pathway is activated through the IKKα/β/γ complex, while the noncanonical pathway involves NIK–mediated activation of IKKα homodimers. In the current study, we have investigated the relative roles of these two pathways in hypoxia-dependent NF-κB activation. Lymphotoxin α1β2 (LTα1β2) activated both the canonical and noncanonical NF-κB signaling pathways in HeLa cells. Sustained hypoxia enhanced basal and LTα1β2-induced NF-κB activity in a manner that was dependent upon the canonical but not the noncanonical signaling pathway. Intermittent hypoxia activated NF-κB in a manner that was also primarily dependent upon the canonical pathway. Knockdown of the p65 subunit of the canonical NF-κB pathway was sufficient to abolish the effects of hypoxia on LTα1β2–induced NF-κB activity. Furthermore, in synovial biopsies obtained at arthroscopy from patients with active inflammatory arthritis, the canonical pathway was preferentially activated in those patents with lower joint pO₂ values. In summary, we hypothesize that hypoxia enhances NF-κB activity primarily through affecting the canonical pathway. Antioxid. Redox Signal. 11, 2057–2064.