AbstractNF‐κB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK‐1, IKK‐2, and NEMO/IKKγ, it is the interaction between IKK‐2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C‐terminal LDWSWL residues of IKK‐2 (called the Nemo Binding Domain (NBD)) and the N‐terminus of NEMO. In an effort to characterize the IKK‐2:NEMO interaction, we have investigated several NBD‐containing peptides for their ability to bind NEMO and inhibit the critical IKK‐2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK‐2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD‐containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD‐containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO. Copyright © 2006 John Wiley & Sons, Ltd.