AbstractTNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF‐treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF‐induced ROS as compared to pro‐oxidant‐induced ROS we conclude that TNF‐mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron‐transport inhibitors, glutathione and thiol‐reactive agents but also caspase inhibitors modulate TNF‐induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF‐induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.