Estrogens play a significant role in the development, growth, invasion and metastasis of ovarian tumors. The transcriptional program regulated by 17beta-estradiol (E(2)) in human ovarian cancer cell lines was analyzed using cDNA microarrays containing 1200 cancer-related genes. Twenty-eight transcripts had at least a threefold change in expression in E(2)-treated PEO1 ovarian carcinoma cells compared with controls. These differences were confirmed by real-time quantitative PCR and shown to be dependent upon the expression of functional estrogen receptor-alpha (ERalpha). Consistent with this, these gene expression changes were blocked by the anti-estrogen tamoxifen. The use of ERalpha- and ERbeta-specific ligands allowed molecular dissection of the E(2) response and showed that ERalpha activation was responsible for the observed changes in gene expression, whereas ERbeta played no significant role. Inhibition of de novo protein synthesis by cycloheximide was used to distinguish between primary and secondary target genes regulated by E(2). Actinomycin D was used to show that changes in gene expression levels induced by E(2) were a result of changes in transcription and not due to changes in mRNA stability. The results presented here demonstrate that estrogen-driven growth of epithelial ovarian carcinoma is mediated by activation of ERalpha-mediated, and not ERbeta-mediated, transcription.