Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor κB (NF-κB). Associated with its inhibitor, IκB, NF-κB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, IκB is proteolyzed and NF-κB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to IκB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-κB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, IκBα is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in IκBα nor a mutation in p50 or relA, the two major subunits of NF-κB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of IκBα on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce IκBα degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-κB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other IκB molecules, IκBβ and the recently identified IκBε, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-κB. In addition, this analysis suggests a common step in the signaling pathways that trigger IκBα, IκBβ, and IκBε degradation.