The role of estrogen exposure in breast cancer risk is well-documented, and both estrogen synthesis and actions through the estrogen receptor (ER) have been targeted by therapies to control hormone-dependent breast cancer. The discovery of a second ER form and its therapeutic implications sparked great interest. Both the original ERalpha and the more recently identified ERbeta subtypes bind and respond similarly to many physiological and pharmacological ligands. However, differences in phytoestrogen binding have been noted, and subtype-specific ligands have been developed. Cell-based assays show that ERbeta and its variants are generally less active on gene transcription than ERalpha, and may influence ERalpha activity; however, both gene- and cell-specific responses occur, and nongenomic activities are less well explored. Specific ligands, and methods to disrupt or eliminate receptor subtype expression in animal and cell models, demonstrate that the ERs have both overlapping and distinct biological functions. Overall, in cell-based studies, ERalpha appears to play a predominant role in cell proliferation, and ERbeta is suggested to be antiproliferative. The potential for distinct populations of breast tumors to be identified based on ER subtype expression, and to exhibit distinct clinical behaviors, is of greatest interest. Several studies suggest that the majority of ER-positive tumors contain both subtypes, but that some tumors contain only ERbeta and may have distinct clinical behaviors and responses. Expression of ERbeta together with ERalpha favors positive responses to endocrine therapy in most studies, and additional studies to determine if the addition of ERbeta to ERalpha as a tumor marker is of clinical benefit are warranted. In contrast, the positive association between ERbeta and HER2 expression in high-grade ERalpha-negative breast cancer does not favor positive responses to endocrine therapy. Expression of ERbeta in specific clinical subpopulations, and the potential for therapies targeting ERbeta specifically, is discussed.