The emerging cancer stem cell (CSC)/ tumor-initiating cell (TIC) hypothesis seeks to explain cancer persistence and recurrence. It supposes that a small subgroup of cells within tumors is multipotent and has chemoresistant features, which contributes to the heterogeneous and regenerative phenotype of recurring tumors. In breast cancer, previous studies have shown the TIC subgroup can be propagated in-vitro from both primary tumors and established cell lines by culturing the cells as “mammospheres”. The goal of this project is to characterize the antiestrogen response of mammospheres (MCF7S) derived from estrogen receptor (ERα)-positive mammary epithelial MCF7 cells, and to determine if there is enrichment of putative TICs after antiestrogen challenge. It is shown here that MCF7S cell proliferation was decreased by antiestrogen treatments. Sphere formation was affected by a selective estrogen receptor modulator (SERM), but not by a selective estrogen receptor down-regulator (SERD). Using the sphere formation assay, we determined that the sphere forming ability returned after drug removal and that there was no significant decrease in sphere formation frequency. Surprisingly, stable ERα knockdown MCF7S cells were more sensitive to SERM and exhibited diminished sphere formation frequency. Treatment of ERα knockdown cells with SERD did not significantly change cell proliferation, sphere formation or frequency. In summary, the data showed that a stable fraction of potential TICs remained after antiestrogen challenge. The results also demonstrated that ERα is not essential for MCF7S survival, but may mediate antiestrogen function. Further studies on SERMs may be warranted as SERMs may target potential TICs that use alternative mitogenic pathways.