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-kappaB, a master regulator of genes involved in innate immunity, inflammation, and apoptosis. NF-kappaB can be activated through two distinct signaling pathways termed the canonical and noncanonical pathways, respectively. The canonical pathway is activated through the IKKalpha/beta/gamma complex, while the noncanonical pathway involves NIK-mediated activation of IKKalpha homodimers. In the current study, we have investigated the relative roles of these two pathways in hypoxia-dependent NF-kappaB activation. Lymphotoxin alpha1beta2 (LTalpha1beta2) activated both the canonical and noncanonical NF-kappaB signaling pathways in HeLa cells. Sustained hypoxia enhanced basal and LTalpha1beta2-induced NF-kappaB activity in a manner that was dependent upon the canonical but not the noncanonical signaling pathway. Intermittent hypoxia activated NF-kappaB in a manner that was also primarily dependent upon the canonical pathway. Knockdown of the p65 subunit of the canonical NF-kappaB pathway was sufficient to abolish the effects of hypoxia on LTalpha1beta2-induced NF-kappaB 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-kappaB activity primarily through affecting the canonical pathway.