Expression of estrogen receptor β (ERβ) protein in human breast cancer and correlation with clinicopathological factors have been reported by many investigators, but many of them used ERβ antibodies that react with both wild-type ERβ (ERβwt) and splicing variant isoform. Therefore, the frequency and correlation with clinicopathological factors of ERβwt expression remain to be established. In the present study a monoclonal antibody EMR02, specific for ERβwt, was used in formalin-fixed paraffin-embedded sections from 225 female primary breast cancer patients diagnosed as having invasive ductal carcinoma. Expression of ERα, progesterone receptor (PgR) and HER2/neu were also investigated by immunohistochemistry. For ERβwt, ERα and PgR, positivity was defined as nuclear staining in >10% of the cancer cells. HER2/neu overexpression was defined as a Hercep test score 3+. Positivity for ERβwt, ERα, PgR and HER2/neu overexpression was 55%, 74%, 61% and 25%, respectively. The expression of ERβwt had a positive correlation with ERα (P = 0.018) and PgR (P = 0.02). There was significant positive correlation between ERβwt expression and HER2/neu overexpression (P < 0.0001). According to multivariate logistic regression analysis the most significant association was between ERβwt expression and HER2/neu overexpression (P < 0.0001). These results suggest that clinical significances of ERβwt expression in human breast cancer patients may be more complex.