Leucine zipper‐bearing kinase (LZK) is a novel member of the mixed lineage kinase (MLK) family [Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J. P., and Kawasaki, T. (1997) J. Biol. Chem.272, 28622–28629]. We have previously shown that LZK activates the c‐Jun‐NH₂ terminal kinase (JNK) pathway, but not the extracellular signal‐related kinase (ERK) pathway, by acting as a mitogen‐activated protein kinase kinase kinase (MAPKKK) [Ikeda, A., Hasegawa, K., Masaki, M., Moriguchi, T., Nishida, E., Kozutsumi, Y., Oka, S., and Kawasaki, T. (2001) J. Biochem.130, 773–781]. However, the mode of activation of LZK remains largely unknown. By means of a yeast two‐hybrid screening system, we have identified a molecule localized to mitochondria, antioxidant protein‐1 (AOP‐1), that binds to LZK and which acts as a modulator of LZK activity. Recently, several MAPKKKs involved in the JNK pathway, such as MEKK1, TAK1 and MLK3, were shown, using over‐expression assay systems, to activate a transcription factor, NF‐κB, through activation of the IKK complex. Using similar assay systems, we demonstrated that LZK activated NF‐κB‐dependent transcription through IKK activation only weakly, but this was reproducible, and that AOP‐1 enhanced the LZK‐induced NF‐κB activation. We also provided evidence that LZK was associated directly with the IKK complex through the kinase domain, and that AOP‐1 was recruited to the IKK complex through the binding to LZK.