Reactive oxygen species are thought to be messengers for nuclear factor (NF)-kappa B activation because its activation can be abrogated by antioxidants. However, this study identifies, for the first time, NF-kappa B activators that are insensitive to antioxidants. NF-kappa B activation that is induced by either calyculin A or okadaic acid (inhibitors of serine/threonine protein phosphatases 1 and 2A) is not blocked by N-acetylcysteine or dihydrolipoate in Jurkat and U937 cells. Nonetheless, these antioxidants block induction by TNF-alpha, lymphotoxin, and PMA. Unlike okadaic acid and calyculin A, neither TNF-alpha, lymphotoxin, nor PMA inhibited activities of phosphatases 1 and 2A. NF-kappa B activation induced by okadaic acid or calyculin A was not blocked by a myosin light chain kinase inhibitor, but was prevented by a protease inhibitor. The mitochondrial inhibitor, rotenone, also inhibited NF-kappa B activation by calyculin A; however, this inhibition was accompanied by a depletion of cellular ATP. These results suggest that 1) phosphatase inhibitors either target a component of signal transduction, which occurs downstream to an antioxidant-sensitive step or use distinct signaling pathways; 2) inhibition of phosphatases 1 and 2A is not a step in the pathway of TNF-alpha-, lymphotoxin-, or PMA-induced NF-kappa B activation; 3) myosin light chain kinase does not participate in NF-kappa B activation; and 4) activation of NF-kappa B by phosphatase inhibitors is controlled by proteases.