Normal fibroblasts are resistant to the cytotoxic activity of tumor necrosis factor-alpha, but are rendered TNF-sensitive upon oncogenic expression of c-Myc. Free radical generation has been implicated in non-cytotoxic TNF-signaling but also as a mediator of TNF-induced cell death. In this study we used Rat1 fibroblasts containing a conditionally active form of oncogenic c-Myc (MycER) to investigate single cell line TNF-induced free oxygen radical formation during the non-cytotoxic TNF-response (inactive c-Myc) and cytotoxic response (active c-Myc). The generation of reactive oxygen species (ROS) was assayed using a fluorescent probe, dichlorodihydrofluorescein (DCFH-DA), and the following cellular injury by measuring the high energy nucleotide (ATP, ADP and AMP) depletion. We found that TNF treatment of Rat1 cells containing c-Myc in an inactive form did not induce a detectable level of ROS generation. In contrast, TNF treatment of Rat1 cells containing activated c-Myc caused fluorescence reaction indicative of ROS generation within 80 min after DCFH-DA exposure of the cells. The nucleotide depletion likely reflected the action of ROS, since the nucleotide depletion caused by TNF or oxidants such as menadione or H2O2 in cells with active c-Myc was partly inhibited by the anti-oxidant N-acetylcysteine.