Non-alcoholic steatohepatitis (NASH) develops in a subset of patients with non-alcoholic fatty liver disease (NAFLD), but the exact mechanisms involved in the progression of NAFLD to NASH remain poorly understood. We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in the apoptosis of hepatocytes that is related to the severity of NASH. We separated primary hepatocytes from the NAFLD liver caused by a high-fat diet. The production of intracellular reactive oxygen species was increased in steatotic hepatocytes, which were also sensitive to TNF-alpha. This factor induced significant apoptosis through the signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK) pathway. We describe here a novel culture model of steatotic hepatocytes separated from the NAFLD liver, and demonstrate that TNF-alpha induces their apoptosis in vitro. (C) 2009 Elsevier Inc. All rights reserved.