Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of AdSOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth. Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress. Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (iv) BCNU, recapitulating clinical usage, and intratumoral AdMnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results, suggesting the potential use of oxidative stress-induced growth inhibitory treatments for breast cancer patients.