CD40, a cell surface receptor in the tumor necrosis factor receptor family, first identified and functionally characterized on B lymphocytes, is also expressed on epithelial and other cells and is now thought to play a more general role in immune regulation. Overexpression of the NF kappa B activator 1 (Act1) leads to the activation of both NF kappa B and Jun kinase in epithelial cell lines. Endogenous Act1 is recruited to the CD40 receptor in human intestinal (HT29) and cervical (HeLa) epithelial cells upon stimulation with CD40 ligand, indicating that Act1 is involved in this signaling pathway. Act1 also interacts with tumor necrosis factor receptor-associated factor 3, a component involved in CD40-activated pathway. Furthermore, transfection of Act1 into C33A cervical epithelial cells, which do not express it, renders these cells sensitive to CD40 ligand-induced NF kappa B activation and protects them from CD40 ligand-induced apoptosis. We conclude that Act1 plays an important role in CD40-mediated signaling in epithelial cells.