The role of estrogen (ER) and progesterone receptors (PR) in breast cancer is well established. Identification of the second human estrogen receptor, the estrogen receptor beta (ERbeta), prompted us to evaluate its role in breast cancer. We studied the expression of ERbeta by immunohistochemistry and mRNA in situ hybridization in 92 primary breast cancers and studied its association with ERalpha, PR, and various other clinicopathological factors. Sixty percent of tumors were defined as ERbeta-positive (nuclear staining in >20% of the cancer cells). Normal ductal epithelium and 5 of 7 intraductal cancers were also found to express ERbeta. Three-fourths of the ERalpha- and PR-positive tumors were positive for ERbeta, whereas ERalpha and PR were positive in 87% and 67% of ERbeta-positive tumors, respectively. ERbeta was associated with negative axillary node status (P < 0.0001), low grade (P = 0.0003), low S-phase fraction (P = 0.0003), and premenopausal status (P = 0.04). In conclusion, the coexpression of ERbeta with ERalpha and PR as well as its association with the other indicators of low biological aggressiveness of breast cancer suggest that ERbeta-positive tumors are likely to respond to hormonal therapy. The independent predictive value of ERbeta remains to be established.