Activation of the transcription factor NF-kappaB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-kappaB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-kappaB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-kappaB activation. CHUK associates with the NF-kappaB inhibitory protein, IkappaB-alpha, in mammalian cells. CHUK specifically phosphorylates IkappaB-alpha on both serine 32 and serine 36, modifications that are required for targeted degradation of IkappaB-alpha via the ubiquitin-proteasome pathway. This phosphorylation of IkappaB-alpha is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IkappaB-alpha kinase that links TNF- and IL-1-induced kinase cascades to NF-kappaB activation.