Background: Reactive oxygen species (ROS) are not only harmful by-products of the cellular metabolism but also essential components of cell signaling, contributing to various physiologic features including cytokine and growth factor signaling. Here, we examined the role of ROS in the cytotoxic effects of tumor necrosis factor-α (TNF-agr;) in MCF-7 cells and their drug-resistant counterparts, MCF-7/MX cells. Materials and Methods: ROS levels were evaluated following TNF-agr; exposure using 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) as fluorescent probe, and the TNF-agr; cytotoxic effects were examined using the dimethylthiazolyl-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: TNF-agr; led to ROS accumulation only in MCF-7/MX and not in MCF-7 cells. The role of ROS in the cytotoxic effects of TNF-agr; was further evaluated by inhibition of ROS accumulation in MCF-7/MX cells and by induction of ROS generation in MCF-7 cells along with TNF-agr; treatment. ROS accumulation sensitized the MCF-7 cells to the cytotoxic effects of TNF-agr; while inhibition of ROS accumulation attenuated the cytotoxic effects of TNF-agr; in MCF-7/MX cells. Following TNF-agr; treatment, the activities of antioxidant enzymes (superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase) were evaluated in both cell lines. The results of the enzyme assays revealed that superoxide dismutase activity was enhanced in MCF-7 but not in MCF-7/MX cells. Conclusions: ROS accumulation in MCF-7/MX cells may be involved in the higher cytotoxic effects of TNF-agr; in the MCF-7/MX cell line.