Nitrofurazone, a veterinary antimicrobial drug, causes mammary and ovarian tumors in animals. We investigated the mechanisms of carcinogenesis by nitrofurazone. Nitrofurazone significantly stimulated the proliferation of estrogen-dependent MCF-7 cells. Nitrofurazone caused Cu(II)-mediated damage to 32 P-5′-end-labeled DNA fragments obtained from human genes only when cytochrome P450 reductase was added. DNA damage was inhibited by catalase and bathocuproine. DNA damage was preferably induced at the 5′-ACG-3′ sequence, a hotspot of the p53 gene. These findings suggest that nitrofurazone metabolites are involved in tumor initiation through oxidative DNA damage and nitrofurazone itself enhances cell proliferation, leading to promotion and/or progression in carcinogenesis.