We describe a new signal mediator of NF-kappaB activation, TANK, that acts in a pathway common to two surface receptors CD40 and TNFR II. TRAF family members interact directly with these receptors. Using the yeast two-hybrid system, TANK was identified as an intracellular protein without previous homologs that interacts with all three known TRAF family members. In cotransfection experiments, TANK and TRAF2 activate NF-kappaB synergistically, requiring both the amino-terminal portion of TANK and the ring finger domain of TRAF2. TANK has a negatively acting carboxyl terminus and is constitutively inactive, but TRAF2 binding overcomes the internal inhibitory influence. We propose that ligand binding to CD40 or TNFR II leads to the formation of a TRAF2/TANK complex, mediating NF-kappaB activation.