Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) has mainly been involved in signaling from CD40 and IL-1 receptor family. While TNF-alpha exerts various biological effects including cell death, the role of TRAF6 in the TNF-alpha signaling remains to be unclear. Here, we demonstrated that murine embryonic fibroblasts (MEFs) derived from TRAF6 knockout (TRAF6KO) mice have increased sensitivity to actinomycin D plus TNF-alpha-induced cell death compared with wild-type MEF. Reactive oxygen species (ROS) were accumulated more in TRAF6KO MEF than in wild-type MEF. An antioxidant, butylated hydroxyanisole (BHA) completely inhibited TNF-alpha-induced cell death and DNA fragmentation. Thus, the TNF-alpha-induced cell death in TRAF6KO MEF was ROS-dependent. Reconstitution of full-length TRAF6 but not N-terminal-deleted TRAF6 constructs in TRAF6KO MEF reversed TNF-alpha-induced cell death, ROS accumulation, and DNA fragmentation completely. Thus, we concluded that resistance against TNF-alpha-induced cell death is rendered by TRAF6, which regulates ROS accumulation. (c) 2006 Elsevier Inc. All rights reserved.