Initiation of the genetic programs for inflammation and immunity involves nuclear mobilization of transcription factor NF-kappaB. This signal-dependent process is controlled in part by the beta-catalytic subunit of IkappaB kinase (IKKbeta), which marks IkappaBalpha and other cytoplasmic inhibitors of NF-kappaB for proteolytic destruction. The catalytic activity of IKKbeta is stimulated by pathologic and physiologic inducers of NF-kappaB, such as the Tax oncoprotein and proinflammatory cytokines. We now report evidence that these NF-kappaB inducers target IKKbeta for conjugation to ubiquitin (Ub) in mammalian cells. The apparent molecular size of modified IKKbeta is compatible with monoubiquitination rather than attachment of a multimeric Ub chain. The modification is contingent upon signal-induced phosphorylation of the activation T loop in IKKbeta at Ser-177/Ser-181. The formation of IKKbeta-Ub conjugates is disrupted in cells expressing YopJ, a Ub-like protein protease that interferes with the NF-kappaB signaling pathway. These findings indicate an important mechanistic link between phosphorylation, ubiquitination, and the biologic action of IKKbeta.