Pro-inflammatory cytokines activate the transcription factor NF-κB by stimulating the activity of a protein kinase that phosphorylates IκB, an inhibitor of NF-κB^,,,,, at sites that trigger its ubiquitination and degradation. This results in the nuclear translocation of freed NF-κB dimers and the activation of transcription of target genes,. Many of these target genes code for immunoregulatory proteins,. A large, cytokine-responsive IκB kinase (IKK) complex has been purified and the genes encoding two of its subunits have been cloned,,. These subunits, IKK-α and IKK-β, are protein kinases whose function is needed for NF-κB activation by pro-inflammatory stimuli. Here, by using a monoclonal antibody against IKK-α, we purify the IKK complex to homogeneity from human cell lines. We find that IKK is composed of similar amounts of IKK-α, IKK-β and two other polypeptides, for which we obtained partial sequences. These polypeptides are differentially processed forms of a third subunit, IKK-γ. Molecular cloning and sequencing indicate that IKK-γ is composed of several potential coiled-coil motifs. IKK-γ interacts preferentially with IKK-β and is required for the activation of the IKK complex. An IKK-γ carboxy-terminal truncation mutant that still binds IKK-β blocks the activation of IKK and NF-κB.