The nuclear factor-kappaB (NF-kappaB) transcription factors control many physiological processes including inflammation, immunity, and apoptosis. In our search for NF-kappaB inhibitors from natural resources, we identified yangonin from Piper methysticum as an inhibitor of NF-kappaB activation. In the present study, we demonstrate that yangonin potently inhibits NF-kappaB activation through suppression of the transcriptional activity of the RelA/p65 subunit of NF-kappaB. This compound significantly inhibited the induced expression of the NF-kappaB-reporter gene. However, this compound did not interfere with tumor necrosis factor-alpha (TNF-alpha)-induced inhibitor of kappaBalpha (IkappaBalpha) degradation, p65 nuclear translocation, and DNA-binding activity of NF-kappaB. Further analysis revealed that yangonin inhibited not only the induced NF-kappaB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Moreover, yangonin did not inhibit TNF-alpha-induced activation of p38, but it significantly impaired activation of extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase. We also demonstrated that pretreatment of cells with this compound prevented TNF-alpha-induced expression of NF-kappaB target genes, such as interleukin 6, interleukin 8, monocyte chemotactic protein 1, cyclooxygenase-2 and inducible nitric oxide. Taken together, yangonin could be a valuable candidate for the intervention of NF-kappaB-dependent pathological conditions such as inflammation.