The majority of breast cancer deaths are because of ineffective treatment of metastatic disease. We previously identified a subpopulation of cells in human breast cancer cell lines that demonstrate high activity of aldehyde dehydrogenase (ALDH) and high expression of CD44. These ALDH(hi)CD44(+) cells displayed enhanced metastatic behavior in vitro and in vivo relative to ALDH(low)CD44(-) cells. The goal of this study was to test the hypothesis that ALDH(hi)CD44(+) breast cancer cells are more resistant to standard cancer therapy, and that inhibiting ALDH activity through all-trans retinoic acid (ATRA) or the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) sensitizes these cells to treatment. ALDH(hi)CD44(+) and ALDH(low)CD44(-) populations were isolated from MDA-MB-231 and MDA-MB-468 cells lines and exposed to chemotherapy (doxorubicin/paclitaxel) or radiotherapy ± ATRA or DEAB. Cell populations were assessed for differences in survival, colony formation, and protein expression related to therapy resistance and differentiation. Significantly more ALDH(hi)CD44(+) cells survived chemotherapy/radiotherapy relative to ALDH(low)CD44(-) cells (P < 0.001). Glutathione-S-transferase pi, p-glycoprotein, and/or CHK1 were overexpressed in ALDH(hi)CD44(+) populations compared with ALDH(low)CD44(-) populations (P < 0.05). Pre-treatment of cell populations with DEAB or ATRA had no effect on ALDH(low)CD44(-) cells, but resulted in significant initial sensitization of ALDH(hi)CD44(+) cells to chemotherapy/radiotherapy. However, only DEAB had a long-term effect, resulting in reduced colony formation (P < 0.01). ATRA also significantly increased expression of CK8/18/19 in MDA-MB-468 ALDH(hi)CD44(+) cells compared with control (P < 0.05). Our novel findings indicate that ALDH(hi)CD44(+) breast cancer cells contribute to both chemotherapy and radiation resistance and suggest a much broader role for ALDH in treatment response than previously reported.