Overexpression of manganese superoxide dismutase (MnSOD) can sensitize a variety of cancer cell lines to many anticancer drugs. Recent work has shown that cancer cells can be sensitized to cell killing by raising peroxide levels through increased manganese superoxide dismutase (MnSOD) when combined with inhibition of peroxide removal. Here we utilize the mechanistic property of one such anticancer drug, BCNU, which inhibits glutathione reductase (GR), compromising the glutathione peroxidase system thereby inhibiting peroxide removal. The purpose of this study was to determine if anticancer modalities known to produce superoxide radicals can increase the antitumor effect of MnSOD overexpression when combined with BCNU. To enhance MnSOD, an adenoviral construct containing the cDNA for MnSOD (AdMnSOD) was introduced into human breast cancer cell line, ZR-75-1. AdMnSOD infection alone did not alter cell killing, however when GR was inhibited with either BCNU or siRNA, cytotoxicity increased. Futhermore, when the AdMnSOD + BCNU treatment was combined with agents that enhance steady-state levels of superoxide (TNF-α, antimycin, adriamycin, photosensitizers, and ionizing radiation), both cell cytotoxicity and intracellular peroxide levels increased. These results suggest that the anticancer effect of AdMnSOD combined with BCNU can be enhanced by agents that increase generation of superoxide.