We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-κB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1β (IL-1β) led to ROI production and NF-κB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-κB activation in these cells. IL-1β stimulation of epithelial cells did not generate any ROIs and NF-κB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-κB activation. In monocytic cells, IL-1β treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-κB activation by IL-1β in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-κB activation by IL-1β: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.