While apoptosis has been considered to be identical to programmed cell death, necroptosis, which is morphologically related to necrosis, has emerged as a novel type of programmed cell death. Necroptosis depends on two structurally related kinases, receptor-interacting serine-threonine kinase (RIPK)1 and RIPK3. RIPK1 is activated through oligomerization of upstream adaptor molecules such as Fas-associated protein with death domain (FADD) and TNF receptor-associated death domain (TRADD) that are triggered by TNF alpha or Fas ligand. Activated RIPK1 subsequently interacts with and activates RIPK3, resulting in necroptosis. However, contribution of oxidative stress to execution of necroptosis is still controversial. We found that a selective inhibitor for RIPK1, necrostatin-1 (Nec-1) significantly blocked TNF alpha-induced cell death and ROS accumulation in NF-kappa B activation-deficient cells. This suggests that these cells mostly died by necroptosis upon TNF alpha stimulation. Intriguingly, an antioxidant, butylated hydroxyanisole (BHA) blocked TNF alpha-induced necroptosis and ROS accumulation in NF-kappa B activation-deficient cells. However, Nec-1, but not BHA, inhibited TNF alpha-induced phosphorylation of RIPK1 in these cells, suggesting that ROS play a crucial role in execution of necroptosis downstream of RIPK1 activation. Structural and functional analyses using BHA related compounds revealed that both tert-butyl and hydroxy groups of BHA are crucial for its anti-necroptotic function. Together, these results suggest that TNF alpha-induced necroptosis is tightly associated with oxidative stress, and oxidative stress is induced downstream of RIPK1 activation. (C) 2013 Elsevier Inc. All rights reserved.