Activation of the transcription factor nuclear factor kappa B (NF-κB) by inflammatory cytokines requires the successive action of NF-κB–inducing kinase (NIK) and IκB kinase–α (IKK-α). A widely expressed protein kinase was identified that is 52 percent identical to IKK-α. IκB kinase–β (IKK-β) activated NF-κB when overexpressed and phosphorylated serine residues 32 and 36 of IκB-α and serines 19 and 23 of IκB-β. The activity of IKK-β was stimulated by tumor necrosis factor and interleukin-1 treatment. IKK-α and IKK-β formed heterodimers that interacted with NIK. Overexpression of a catalytically inactive form of IKK-β blocked cytokine-induced NF-κB activation. Thus, an active IκB kinase complex may require three distinct protein kinases.