Activation of a large multisubunit protein kinase, called the inhibitor κB kinase (IKK) complex, is central to the induction of the family of transcription factors nuclear factor κB. IKK is comprised of two catalytic subunits, IKKα and IKKβ, and a regulatory IKKγ subunit. It is known that the catalytic IKKβ and regulatory IKKγ subunits associate through interactions mediated by the N-terminal region of IKKγ and an 11-mer peptide located near the C-terminus of IKKβ. In this study, we have defined the minimal IKKγ segment that binds IKKβ and determined the binding affinity of the IKKβ/IKKγ complex. We identified that the N-terminal segment spanning residues 40−130 of IKKγ binds the IKKβ C-terminal domain (residues 665−756) with Kd ≈ 25 nM. Several smaller N-terminal IKKγ deletion mutants within the N-terminal 130 residues, although in some cases retained IKKβ binding activity, showed a tendency to aggregate and formed covalently linked complexes. However, expansion of the C-terminus of these fragments to re...