The effects of estrogen on gene expression in mammary cells are mediated by interaction of the estrogen receptor (ER) with estrogen response elements (EREs) in target DNA. While the ER is the primary initiator of transcription, the recruitment of coregulatory proteins to the DNA-bound receptor influences estrogen-responsiveness. To better understand how estrogen alters gene expression, we identified proteins associated with the DNA-bound ER. Surprisingly, the antioxidant enzyme Cu/Zn superoxide dismutase (SOD1), which is known primarily as a scavenger of superoxide, was associated with the DNA-bound receptor. We have now demonstrated that SOD1 interacts with ER from MCF-7 cell nuclear extracts and with purified ER and that SOD1 enhances binding of ER to ERE-containing DNA. Although SOD1 decreases transcription of an estrogen-responsive reporter plasmid in transiently transfected U2 osteosarcoma cells, RNA interference assays demonstrate that SOD1 is required for effective estrogen responsiveness of the endogenous pS2, progesterone receptor, cyclin D1, and cathepsin D genes in MCF-7 breast cancer cells. Furthermore, ER and SOD1 are associated with regions of the pS2 and PR genes involved in conferring estrogen-responsive gene expression. Interestingly, when MCF-7 cells are exposed to 17-estradiol (E₂) and superoxide generated by addition of potassium superoxide (KO₂) to the cell medium, SOD1 levels are increased and tyrosine nitration, which is an indicator of oxidative stress-induced protein damage, is significantly diminished. Our studies have identified a new role for SOD1 in regulating estrogen-responsive gene expression and suggest that the E₂- and KO₂-induced increase in SOD1 may play a role in the survival of breast cancer cells and the progression of mammary tumors.