The murine fibrosarcoma cell line WEHI 164 is well known for its susceptibility to tumor necrosis factor (TNFα). We have studied the activation of the transcription factor NF-κB when WEHI 164 cells are challenged with TNFα. NF-κB is retained in the cytoplasm of unchallenged cells by its inhibitor IκB-α. Upon cellular stimulation, IκB-α is functionally inactivated and NF-κB translocates to the nucleus. The extent of the cytotoxic effect and that of nuclear translocation of NF-κB show the same TNFα dependence. TNFα induces a rapid and transient activation of NF-κB in WEHI 164 cells which is followed by a second, long lasting phase in which the amount of NF-κB complex in the nucleus remains at about 50% of maximum. Upon TNFα treatment, IκB-α is rapidly degraded. However, newly synthesized IκB-α can be demonstrated later in the cell cytosol. A persistent nuclear localization of NF-κB is an obligatory step for the cytotoxic effect to take place. Thus, WEHI 164 cells treated with TNFα for up to 6 h can be rescued as long as NF-κB relocalizes to the cytoplasm in its inactive form. On the other hand, TNFα treatments as short as 15 min cause the cytotoxic effect provided that NF-κB remains in the nucleus. The activation of NF-κB is controlled by both phosphorylation and proteolysis. The activation of NF-κB can be blocked by the cysteine protease inhibitor calpain inhibitor I and the serine protease inhibitor TPCK. Signal-induced phosphorylation of IκB-α does not lead to the dissociation of the inhibitor from NF-κB. Phosphorylation appears to regulate the inhibitory activity of IκB-α both positively and negatively, since inhibitors of protein kinases have opposite effects. Thus, treatment of cells with staurosporin induced a partial activation of NF-κB and was synergistic with TNFα-induced activation. Calphostin C, on the other hand, can block the activation of NF-κB by TNFα, also blocking its proteolytic degradation.