NEMO is an essential regulatory component of the I kappa B kinase (IKK) complex, which controls activation of the NF-kappa B signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (14202) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H2O2 decreases TNF alpha-induced IKK activity in NEMO-reconstituted cells, and that TNF alpha has a diminished ability to activate NF-kappa B DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein. (C) 2007 Elsevier Inc. All rights reserved.