Tumor necrosis factor (TNF) has been shown to be cytotoxic to tumor cell lines in vitro, but the mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we used various inhibitors to investigate the sequence of events that lead to cytotoxic effects of TNF on L.P3 cells, a highly sensitive, murine fibroblast cell line. Our results indicate that mitochondrial respiration chains are damaged by a hydroxyl radical at an early stage of the cell lysis after TNF treatment. This event is followed by the activation of phospholipase A2, and finally leads to cell lysis.