The transcription factor NF-kappa B is a key regulator of the cellular inflammatory and immune response. Therefore, components of the NF-kappa B-activating signaling pathways are frequent targets for antiinflammatory agents. This study shows that the sesquiterpene lactone parthenolide inhibits a common step in NF-kappa B activation by preventing the TNF-alpha-induced induction of I kappa B kinase (IKK) and IKK beta, without affecting the activation of p38 and c-Jun N-terminal kinase. Parthenolide impairs NF-kappa B-dependent transcription triggered by expression of TNFR-associated factor-2, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK1), and NF-kappa B-inducing kinase. This compound also prevents activation of both IKKs and DNA binding of NF-kappa B induced by MEKK and NF-kappa B-inducing kinase. Parthenolide targets a component of the I kappa B kinase complex without directly inhibiting IKK alpha, IKK beta, or MEKK1. Therefore, this sesquiterpene lactone could serve as a lead compound for the development of antiinflammatory remedies and is suitable as a molecular tool, allowing the dissection of TNF-alpha-derived signaling pathways leading to the activation of NF-kappa B, c-Jun N-terminal kinase, and p38.