Estrogens generally stimulate the proliferation of estrogen receptor (ER)-containing breast cancer cells, but they also suppress proliferation of some ER-positive breast tumors. Using a genome-wide analysis of gene expression in two ER-positive human breast cancer cell lines that differ in their proliferative response to estrogen, we sought to identify genes involved in estrogen-regulated cell proliferation. To this end, we compared the transcriptional profiles of MCF-7 and MDA-MB-231ER+ cells, which have directionally opposite estradiol-dependent proliferation patterns, MCF-7 cells being stimulated and 231ER+ cells suppressed by estradiol. We identified a set of ca. 70 genes regulated by estradiol in both cells, with most being oppositely regulated by hormone. Using a variety of bioinformatics approaches, we found the E2F binding site to be over-represented in the potential regulatory regions of many cell-cycle related genes stimulated by estrogen in MCF-7 but inhibited by estrogen in 231ER+ cells. Biochemical analyses confirmed that E2F1 and E2F downstream target genes were increased in MCF-7 and decreased in 231ER+ cells upon estrogen treatment. Furthermore, RNAi-mediated knockdown of E2F1 blocked estrogen regulation of E2F1 target genes and resulted in loss of estrogen regulation of proliferation. These results demonstrate that regulation by estrogen of E2F1, and subsequently its downstream target genes, is critical for hormone regulation of the proliferative program of these breast cancer cells, and that gene expression profiling combined with bioinformatic analyses of transcription factor binding site enrichment in regulated genes can identify key components associated with nuclear receptor hormonal regulation of important cellular functions.