Although it is well established that reactive oxygen intermediates mediate the NF-kappaB activation induced by most agents, how H2O2 activates this transcription factor is not well understood. We found that treatment of human myeloid KBM-5 cells with H2O2 activated NF-kappaB in a dose- and time-dependent manner much as tumor necrosis factor (TNF) did but unlike TNF, H2O2 had no effect on IkappaBalpha degradation. Unexpectedly, however, like TNF-induced activation, H2O2-induced NF-kappaB activation was blocked by the calpain inhibitor N-Ac-Leu-Leu-norleucinal, suggesting that a proteosomal pathway was involved. Although H2O2 activated IkappaBalpha kinase, it did not induce the serine phosphorylation of IkappaBalpha. Like TNF, H2O2 induced the serine phosphorylation of the p65 subunit of NF-kappaB, leading to its nuclear translocation. We found that H2O2 induced the tyrosine phosphorylation of IkappaBalpha, which is needed for NF-kappaB activation. We present several lines of evidence to suggest that the Syk protein-tyrosine kinase is involved in H2O2-induced NF-kappaB activation. First, H2O2 activated Syk in KBM-5 cells; second, H2O2 failed to activate NF-kappaB in cells that do not express Syk protein; third, overexpression of Syk increased H2O2-induced NF-kappaB activation; and fourth, reduction of Syk transcription using small interfering RNA inhibited H2O2-induced NF-kappaB activation. We also showed that Syk induced the tyrosine phosphorylation of IkappaBalpha, which caused the dissociation, phosphorylation, and nuclear translocation of p65. Thus, overall, our results demonstrate that H2O2 induces NF-kappaB activation, not through serine phosphorylation or degradation of IkappaBalpha, but through Syk-mediated tyrosine phosphorylation of IkappaBalpha