Estrogens are known to regulate the proliferation of breast cancer cells and to alter their cytoarchitectural and phenotypic properties, but the gene networks and pathways by which estrogenic hormones regulate these events are only partially understood. We used global gene expression profiling by Affymetrix GeneChip microarray analysis, with quantitative PCR verification in many cases, to identify patterns and time courses of genes that are either stimulated or inhibited by estradiol (E2) in estrogen receptor (ER)-positive MCF-7 human breast cancer cells. Of the >12,000 genes queried, over 400 showed a robust pattern of regulation, and, notably, the majority (70%) were down-regulated. We observed a general up-regulation of positive proliferation regulators, including survivin, multiple growth factors, genes involved in cell cycle progression, and regulatory factor-receptor loops, and the down-regulation of transcriptional repressors, such as Mad4 and JunB, and of antiproliferative and proapoptotic genes, including B cell translocation gene-1 and -2, cyclin G2, BCL-2 antagonist/killer 1, BCL 2-interacting killer, caspase 9, and TGFbeta family growth inhibitory factors. These together likely contribute to the stimulation of proliferation and the suppression of apoptosis by E2 in these cells. Of interest, E2 appeared to modulate its own activity through the enhanced expression of genes involved in prostaglandin E production and signaling, which could lead to an increase in aromatase expression and E2 production, as well as the decreased expression of several nuclear receptor coactivators that could impact ER activity. Our studies highlight the diverse gene networks and metabolic and cell regulatory pathways through which this hormone operates to achieve its widespread effects on breast cancer cells.