2-Methoxyestradiol (2-ME), a new anticancer agent currently in clinical trials, has been demonstrated to inhibit superoxide dismutase (SOD) and to induce apoptosis in leukemia cells through a free radical–mediated mechanism. Because the accumulation of superoxide (O₂⁻) by inhibition of SOD depends on the cellular generation of O₂⁻, we hypothesized that the endogenous production of superoxide may be a critical factor that affects the antileukemia activity of 2-ME. In the present study, we investigated the relationship between cellular O₂⁻ contents and the cytotoxic activity of 2-ME in primary leukemia cells from 50 patients with chronic lymphocytic leukemia (CLL). Quantitation of O₂⁻ revealed that the basal cellular O₂⁻ contents are heterogeneous among patients with CLL. The O₂⁻ levels were significantly higher in CLL cells from patients with prior chemotherapy. CLL cells with higher basal O₂⁻ contents were more sensitive to 2-ME in vitro than those with lower O₂⁻ contents. There was a significant correlation between the 2-ME–induced O₂⁻increase and the loss of cell viability. Importantly, addition of arsenic trioxide, a compound capable of causing reactive oxygen species (ROS) generation, significantly enhanced the activity of 2-ME, even in the CLL cells that were resistant to 2-ME alone. These results suggest that the cellular generation of O₂⁻ plays an important role in the cytotoxic action of 2-ME and that it is possible to use exogenous ROS-producing agents such as arsenic trioxide in combination with 2-ME to enhance the antileukemia activity and to overcome drug resistance. Such a combination strategy may have potential clinical applications.