Topoisomerase II-alpha (topo II alpha) is the key target enzyme for the topoisomerase inhibitor class of anti-cancer drugs. In normal cells, topo II alpha is expressed predominantly in the S/G2/M phase of the cell cycle. In malignant cells, in vitro studies have indicated that the expression of topo II alpha is both higher and less dependent on proliferation state in the cell. We studied fresh specimens from 50 cases of primary breast cancer. The expression of topo II alpha in different cell cycle phases was analyzed with two-parameter flow cytometry using the monoclonal antibody SWT3D1 and propidium iodide staining. The expression of topo II alpha was significantly higher in the S/G2/M phase of the cell cycle than in the G0/G1 phase in both DNA diploid and DNA non-diploid tumors. In 18 of 21 diploid tumors, and in 25 of 29 non-diploid tumors, >50% of the topo II alpha-positive cells were in the G0/G1 phase. This significant expression of topo II alpha in the G0/G1 phase of the cell cycle may have clinically important implications for treatment efficacy of topoisomerase II inhibitors.