Activation of transcription factor NF-κB involves the signal-dependent degradation of basally phosphorylated inhibitors such as IκBα. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-κB activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H 2 O 2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of IκBα. Indeed, mutation of Ser 32 and Ser 36 blocked IL-1β-or PMA-induced NF-κB activation, but had no effect on its activation by H 2 O 2. Although IκBα was phosphorylated upon exposure to H 2 O 2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-κB activation by H 2 O 2, while it had no effect on activation by IL-1β or PMA-ionomycin. This H 2 O 2-inducible phosphorylation was not dependent on IκB kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5, 6-dichloro-1-β-d-ribofuranosyl-benzimidazole) blocks NF-κB activation. H 2 O 2-induced IκBα phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-κB activation by H 2 O 2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.