Studies on the mechanisms of inducible and constitutive activity of NF- κB transcription factors have been hampered by the lack of appropriate mutant cell lines. We have analyzed the defect in the murine S107 plasmacytoma cell line, which was previously found to lack both constitutive and inducible NF-κB activity. Our analysis shows that these cells bear a specific defect that interferes with NF-κB induction by many diverse stimuli, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, UV light, x-rays, and H2O2. This does not however represent a general signal transduction defect, because AP-1 transcription factors are readily induced by the same stimuli. Phosphatase inhibitors such as okadaic acid as well as calyculin A can efficiently induce NF-κB in S107 cells via a pathway apparently insensitive to the radical scavenger pyrrolidine dithiocarbamate. Furthermore, MEKK1 a protein kinase supposedly induced by some of the above stimuli, is also capable of activating NF-κB. Interestingly, both the potent physiological inducer of NF-κB TNFα as well as endoplasmic reticulum overload can induce NF-κB via a PDTC sensitive pathway. In all cases, DNA- binding NF-κB complexes are comprised predominantly of p50-RelA heterodimers, and NF-κB activation results in the induction of transiently transfected or resident reporter genes. In summary, these results suggest that the pathways for many NF-κB-inducing stimuli converge at a specific junction, and this pivotal step is mutated in the S107 cell line. Yet there are alternative routes bypassing this critical step that also lead to NF-κB induction. These routes utilized by tumor necrosis factor α and endoplasmic reticulum overload are still intact in this cell line.