Retinoic acid (RA) inhibits proliferation of estrogen receptor (ER)-positive human breast cancer cells, but not the growth of ER-negative cells. We have shown previously that ER-positive cells express higher levels of retinoic acid receptor (RAR) α, suggesting that RARα gene expression may be regulated in breast cancer cells by estrogens. We here report that estradiol (E2) increases RARα mRNA in a time- and concentration-dependent manner resulting in a marked increase in RARα protein expression, and present evidence that RARα1 is the only known isoform of RARα regulated by E2 in breast cancer cells. In parallel we demonstrate that ER-positive cells exhibit greater RA sensitivity in the presence of E2, suggesting that E2-induced expression of RARα1 is involved in growth inhibition by RA. To directly investigate the role of RARα1 in RA-mediated growth inhibition, we introduced RARα1 expression vectors into RA-resistant and ER-negative MDA-MB-231 cells. The RARα1-transfected cells were growth inhibited by RA, while mock- and untransfected cells were unresponsive. Together, our data indicate that adequate levels of RARα1, either generated by introduction of expression vectors or endogenously induced by estrogens, are required for growth inhibition of breast cancer cells by RA.