Previous studies have demonstrated that peptides corresponding to a six-amino-acid NEMO-binding domain from the C terminus of IκB kinase alpha (IKKα) and IKKβ can disrupt the IKK complex and block NF-κB activation. We have now mapped and characterized the corresponding amino-terminal IKK-binding domain (IBD) of NEMO. Peptides corresponding to the IBD were efficiently recruited to the IKK complex but displayed only a weak inhibitory potential on cytokine-induced NF-κB activity. This is most likely due to the formation of sodium dodecyl sulfate- and urea-resistant NEMO dimers through a dimerization domain at the amino terminus of NEMO that overlaps with the region responsible for binding to IKKs. Mutational analysis revealed different α-helical subdomains within an amino-terminal coiled-coil region are important for NEMO dimerization and IKKβ binding. Furthermore, NEMO dimerization is required for the tumor necrosis factor alpha-induced NF-κB activation, even when interaction with the IKKs is unaffected. Hence, our data provide novel insights into the role of the amino terminus of NEMO for the architecture of the IKK complex and its activation.