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