Here we report the identification of a novel PMA-inducible IκB kinase complex, distinct from the well-characterized high–molecular weight IκB kinase complex containing IKKα, IKKβ, and IKKγ. We have characterized one kinase from this complex, which we designate IKKε. Although recombinant IKKε directly phosphorylates only serine 36 of IκBα, the PMA-activated endogenous IKKε complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKε blocks induction of NF-κB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-κB by TNFα or IL-1. These observations indicate that the activation of NF-κB requires multiple distinct IκB kinase complexes, which respond to both overlapping and discrete signaling pathways.