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