The estrogen receptor (ER)-beta isoform has been recently identified to be distinct from ERalpha isoform and regulates separate sets of genes, and can exert opposite signaling functions depending on the ligand and response elements. Previous studies of ERbeta have been at the mRNA level and few by immunohistochemistry, and the results are inconsistent. In this study the authors compared expression of ERbeta with those of other prognostic biomarkers by immunohistochemistry on tissue microarray slides, and with morphologic parameters on 147 cases of primary breast cancer. Immunoreactivity of more than 10% of cancer cells was considered to be positive. Associations between categoric variables were analyzed using the chi test, and a P value less than 0.05 was considered to be significant. ERbeta was expressed in benign epithelium and stromal cells, and breast cancer cells in 59% of different histologic types of breast cancer. ERbeta was coexpressed with ERalpha in 45% of cases. There was a statistically significant association between expression of ERbeta and Her-2/neu (P<0.000), cathepsin D (P<0.02), p53 (P<0.03), and PS2 (P<0.002). Ki-67 was almost exclusively expressed in ERbeta-positive cells. No statistically significant association was seen between ERbeta expression and histologic grade, DNA ploidy, or S-phase.