Although direct cytotoxicity is a well-established phenomenon of tumor necrosis factor alpha (TNFα)-induced tissue damage, the intracellular events leading to cell death are still poorly understood. To study the cytotoxic effects of TNFα on normal parenchymal cells, rat hepatocytes were purified and incubated with various concentrations of TNFα. Mitochondrial respiration, total protein synthesis, and enzyme release were measured to assess metabolic performance and cell integrity. Treatment with TNFα suppressed mitochondrial respiration in a concentration-dependent fashion, resulting in a reduction of the activity of complex I of the respiratory chain to 67.0 ± 3.5% of that of untreated hepatocytes by 2000 U/mL TNFα. Under these conditions protein synthesis and the release of intracellular enzymes were significantly increased. Both hepatocellular enzyme release and inhibition of mitochondrial respiration appear to be associated with the generation of reactive oxygen intermediates by the hepatocyte itself, because oxygen radical scavengers prevented these adverse effects of TNFα. Inhibition of protein synthesis by cycloheximide as well as addition of cyclic adenosine monophosphate synergistically enhanced the suppression of mitochondrial respiration by TNFα, resulting in complex I activity of 6.9 ± 1.6% and 24.9 ± 2.9% of that of untreated cells. These data indicate that inhibition of mitochondrial respiration is one of the mechanisms by which TNFα induces tissue injury.