Estrogen is converted by cytochrome P450 1B1 to 4-hydroxyestradiol (4-OHE(2)), a putative carcinogenic metabolite of estrogen. This catechol estrogen metabolite is oxidized further to produce a reactive quinone via semiquinone. Redox cycling between 4-OHE(2) and its quinoid generates reactive oxygen species (ROS). ROS not only causes oxidative DNA damage but also promotes neoplastic transformation of initiated cells. In the present study, 4-OHE(2) induced anchorage-independent colony formation in human mammary epithelial cells (MCF-10A). MCF-10A cells treated with 4-OHE(2) exhibited increased accumulation of intracellular ROS. The antioxidant N-acetyl-l-cysteine inhibited the neoplastic transformation induced by 4-OHE(2). ROS overproduced by 4-OHE(2) increased the nuclear translocation of nuclear factor-kappaB (NF-kappaB) and its DNA binding through induction of IkappaB kinase alpha (IKKalpha) and IKKbeta activities. The inhibition of the IKK activities with Bay 11-7082 significantly reduced the anchorage-independent growth induced by 4-OHE(2). The 4-OHE(2)-induced activation of extracellular signal-regulated kinase and Akt resulted in enhanced IKK activities and phosphorylation of IkappaBalpha, thereby inducing NF-kappaB activation and anchorage-independent growth of MCF-10A cells. In conclusion, ROS, concomitantly overproduced during redox cycling of 4-OHE(2), activates IKK signaling, which may contribute to neoplastic transformation of MCF-10A cells.