Nuclear factor kappa B (NF-kappa B)-dependent gene expression plays an important role in numerous cellular processes including stress responses, inflammation and cell proliferation. Therefore, the activity of this transcription factor needs to be tightly regulated. Among others, the NF-kappa B-dependent zinc finger protein A20 is involved in the negative feedback regulation of NF-kappa B activation in response to tumor necrosis factor (TNF). We previously demonstrated that A20 can interact with A20-binding inhibitors of NF-kappa B activation (ABINs), which have the potential to inhibit TNF-induced activation of NF-kappa B upon overexpression. The ABIN proteins were therefore proposed to mediate the NF-kappa B inhibiting function of A20. Here we demonstrate the presence of a short homologous region in ABINs and I kappa B kinase gamma, the regulatory subunit of the I kappa B kinase complex. Site-specific mutagenesis of this region abolished the NF-kappa B inhibiting function of ABIN-1, without affecting the interaction with A20. Furthermore, coexpression of these ABIN-1 mutants interfered in a dominant negative manner with the NF-kappa B inhibiting function of ABIN-1, whereas the A20-mediated inhibition was unaffected. These results suggest that A20 and ABIN-1 probably act independently of their mutual interaction.