Signal-induced activation of the transcription factor NF-κB requires specific phosphorylation of the inhibitor IκBα and its subsequent proteolytic degradation. Phosphorylation of serine residues 32 and 36 targets IκBα to the ubiquitin (Ub)–proteasome pathway. Here we report the identification of a large, multisubunit kinase (molecular mass ∼700 kDa) that phosphorylates IκBα at S32 and S36. Remarkably, the activity of this kinase requires the Ub-activating enzyme (E1), a specific Ub carrier protein (E2) of the Ubc4/Ubc5 family, and Ub. We also show that a ubiquitination event in the kinase complex is a prerequisite for specific phosphorylation of IκBα. Thus, ubiquitination serves a novel regulatory function that does not involve proteolysis.