NF-kappaB is a transcription factor that induces inflammatory cytokines and anti-apoptotic proteins. We designed a new NF-kappaB inhibitor that is based on the structe of the antibiotic epoxyquinomicin C. The designed compound, dehydroxymethyl-epoxyquinomicin (DHMEQ), inhibited the TNF-alpha-induced activation of NF-kappaB, and showed an anti-arthritic effect in mice. Recently, we looked into its mechanism of inhibition. DHMEQ inhibited the TNF-alpha-induced cellular DNA binding of nuclear NF-kappaB, but not the phosphorylation or degradation of I-kappaB. Moreover, DHMEQ inhibited the TNF-alpha-induced nuclear accumulation of p65, a component of NF-kappaB. On the other hand, DHMEQ did not inhibit the nuclear transport of Smad2 and the large T antigen. Also, it did not inhibit the TNF-alpha-induced activation of JNK, but synergistically induced apoptosis with TNF-alpha in human T cell leukemia Jurkat cells. Therefore, DHMEQ specifically inhibited the NF-kappaB-activating pathway in the TNF-alpha-treated cells. Taken together, our data show that DHMEQ is a unique inhibitor of NF-kappaB that acts at the level of the nuclear translocation. It may be useful as an anti-inflammatory and anticancer agent.