Activation of the transcription factor nuclear factor kappa B (NF-kappa B) is controlled by sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit I kappa B. A large multiprotein complex, the I kappa B kinase (IKK) signalsome, was purified from Hela cells and found to contain a cytokine-inducible I kappa B kinase activity that phosphorylates I kappa B-alpha and I kappa B-beta. Two components of the IKK signalsome, IKK-1 and IKK-2, were identified as closely related protein serine kinases containing leucine zipper and helix-loop-helix protein interaction motifs. Mutant versions of IKK-2 had pronounced effects on RelA nuclear translocation and NF-kappa B-dependent reporter activity, consistent with a critical role for the IKK kinases in the NF-kappa B signaling pathway.