The role of reactive oxygen intermediates (ROIs) in nuclear factor-kappaB (NF-kappaB) activation remains a matter of controversy. We have studied whether ROIs played any role in NF-kappaB induction by interleukin-1beta (IL-1beta) in different cell types. Our studies indicated three different pathways. IL-1beta stimulation of lymphoid cells generates ROIs, which are required for IkappaB-alpha degradation and NF-kappaB activation. The source of these ROIs is the 5-lipoxygenase (5-LOX) enzyme. In monocytic cells, ROIs are also produced in response to IL-1beta and necessary for NF-kappaB induction, but their source appears to be the NADPH oxidase complex. Finally, epithelial cells do not generate ROIs after IL-1beta stimulation, but do rapidly activate NF-kappaB. Interestingly, transfection of epithelial cells with the 5-LOX and 5-LOX activating protein expression vectors restored ROI production and ROI-dependent NF-kappaB activation in response to IL-1beta. Our data thus indicate that ROIs are cell type-specific second messengers for NF-kappaB induction by IL-1beta.