Estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines express significantly higher levels of retinoic acid receptor α (RARα) (isoform 1) mRNA than ER-negative HBCs. Estradiol enhances RARα mRNA expression in different ER-positive HBCs by 2–3-fold, which in turn results in increased sensitivity of ER-positive HBCs to the growth inhibitory effects of retinoic acid. To investigate the regulatory mechanisms of estradiol-mediated enhancement of RARα mRNA expression, the functional promoter for the human RARα isoform 1 was cloned and used to assess estradiol-mediated promoter-dependent enhancement of firefly luciferase reporter gene activity in transiently transfected ER-positive (MCF-7 and T47D) and ER-negative (MDA-MB-231) HBCs. Deletional promoter constructs were obtained to further delineate the promoter region responsible for estradiol-mediated enhancement of promoter activity. Here, we present evidence that approximately 130 bp of the promoter fragment preceding the transcriptional start site are responsible for estradiol-mediated enhancement of hRARα gene expression. The estradiol-mediated enhancement is dependent on ER binding. Further deletional analysis showed that a promoter sequence of 42 base pairs, located approximately 100 bases upstream of the transcriptional start site, contains elements for estradiol-mediated enhancement. Specific deletion of either the Sp1 motif or mutations in the imperfect half-palindromic estrogen response element motif of this fragment abolish its estradiol responsiveness in transient transfections.