Topoisomerase IIalpha (topo IIalpha) plays a key role in DNA replication and is a target for multiple chemotherapeutic agents. In breast cancer, topo II expression has been linked to cell proliferation and HER2/neu protein overexpression. However, its relationship with outcome variables is not well established. Formalin-fixed, paraffin-embedded primary breast cancers from 184 women (mean age, 60 years) were stained for topo II by automated immunohistochemistry. A topo II expression index (TI) was determined by counting the number of positive cells per high-power field and calculating an overall mean number of positive cells per high-power field. Tumors with a TI of more than 1 were considered positive, and those with a TI of 1 or less were considered negative. A cell proliferation index was determine d by automated immunohistochemistry using the MIB-1 antibody in an identical technique. HER-2/neu gene amplification (HER-2 amp) was determined by automated fluorescence in situ hybridization using the Ventana unique sequence probe. Fifty-nine (32%) of the tumors had a TI greater than 1. On univariate analysis, increased topo II expression correlated with decreased patient survival (p = .001), advanced tumor stage (p = .034), lymph node metastasis (p = .018), and HER-2 amp (p = .016). Tumor stage (p < .0001), node-positive status (p < .0001), tumor grade (p = .025), HER-2 amp (p < .0001), and MIB-1 overexpression (p = .002) also correlated with survival on univariate analysis. Topo II expression did not correlate with tumor size, grade, estrogen receptor/progesterone receptor status, or disease recurrence. On multivariate analysis, stage (p < .0001), lymph node metastasis (p < .0001), and tumor grade (p = .002) all independently predicted disease-related death. Increased topo II expression is associated with an aggressive form of breast cancer featuring HER-2 amp and predicts disease-related death, lymph node metastasis, and advanced tumor stage.