Phosphorylation of the N‐terminal domain of IκB inhibitory subunits induces activation of the transcription factor NF‐κB. Although serine phosphorylation has been shown to induce ubiquitination and subsequent proteasome‐mediated degradation of IκB‐α, little is known about the mechanisms that lead to release of active NF‐κB in T cells as a consequence of tyrosine phosphorylation of IκB‐α[Imbert, V., Rupec, R.A., Livolsi, A., Pahl, H.L., Traenckner, B.M., Mueller‐Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. & Peyron, J.F. (1996) Cell86, 787–798]. The involvement of the tyrosine kinases p56^lck and ZAP‐70 in this reaction is demonstrated here using specific pharmacological inhibitors and Jurkat mutants unable to express these kinases. Although the inhibitors prevented both pervanadate‐induced phosphorylation of IκB‐α on Tyr42 and NF‐κB activation, we observed that, in p56^lck‐deficient Jurkat mutants, NF‐κB could still associate with IκB‐α despite phosphorylation on Tyr42. Furthermore, the SH2 domain of p56^lck appeared to be required for pervanadate‐induced NF‐κB activation but not for Tyr42 phosphorylation. These results show that p56^lck and ZAP‐70 are key components of the signaling pathway that leads to phosphotyrosine‐dependent NF‐κB activation in T cells and confirm that tyrosine kinases must control at least two different steps to induce activation of NF‐κB. Finally, we show that H₂O₂, which stimulates p56^lck and ZAP‐70 in T cells, is an activator of NF‐κB through tyrosine phosphorylation of IκB‐α.