When human ovarian carcinoma cells are challenged with endotoxin, an I kappa B kinase is transiently induced within 3 to 5 min. This enzyme activity causes the hyperphosphorylation and subsequent degradation of I kappa B which allows NF-kappa B to translocate to the nucleus where it activates transcription. When endotoxin treated cells are rechallenged with a second dose of LPS, I kappa B kinase is not detected and I kappa B remains in the cytoplasm where it sequesters NF-kappa B. We report here the absence of endotoxin inducible I kappa B kinase activity in endotoxin tolerant cells suggesting that I kappa B kinase may play an important role in endotoxin tolerance. When cells tolerant to endotoxin are treated with TNF, I kappa B kinase activity is induced. Thus cells that are endotoxin tolerant are not cross tolerant to TNF. Dexamethasone, a known inhibitor of NF-kappa B activation does not inhibit endotoxin dependent induction of I kappa B kinase suggesting that the mechanism of action of dexamethasone is different from the tolerance mechanism reported here.