Human breast cancer cells (MCF-7) overexpressing manganese-containing superoxide dismutase (MnSOD) by stable or transient transfection were challenged with the cytotoxicity of tumor necrosis factor alpha (TNF-alpha), hyperthermia, and a combination of both. In contrast to the vector control and wild-type MCF-7 cells, the stable MnSOD transfectants showed significant resistance to the cytotoxicity of TNF-alpha (100 units/ml), heat at 43 or 45 degrees C, and a combination of TNF-alpha and heat at 43 degrees C. To probe the correlation of MnSOD levels with cell survival, cell sensitivity to TNF-alpha, heat at 43 degrees C, or a combination of both was also measured after MnSOD cDNA transient transfection. The data showed that the level of cell resistance was proportional to the exogenous MnSOD gene expression. These results suggest that superoxide free radicals or their reaction products are responsible for much of the synergistic cytotoxicity of TNF-alpha and hyperthermia.