Sex steroid hormone receptors play a central role in all stages of prostate cancer. Here, we tested whether estrogen receptor (ER) signaling contributes to telomerase activation, an early event in prostate tumorigenesis. Following 17beta-estradiol (E(2)) treatment, both mRNA encoding the catalytic subunit of human telomerase (hTERT) and telomerase activity were promptly induced in human prostate normal epithelial cells, fresh explants from benign prostate hyperplasia, and prostate cancer explants and cell lines. Reporter expression studies and in vivo chromatin immunoprecipitation assays revealed E(2)-dependent hTERT promoter induction and showed that both ERalpha and ERbeta bound this sequence. Crucially, addition of the anti-estrogen 4-hydroxytamoxifen caused a differential recruitment in vivo of ERalpha and ERbeta onto the hTERT promoter and inhibited telomerase activity. Treatment with the aromatase inhibitor letrozole, which prevented testosterone-mediated interaction between ER and the hTERT estrogen response element, resulted in a negative regulation of telomerase activity. Thus, intracellular conversion of androgens to estrogens may contribute to the etiopathogenesis of prostate cancer. Given the present evidence for direct control of hTERT gene expression and telomerase activity in the prostate by the ER, we suggest that this transcriptional regulator represents a possible therapeutic target in prostate cancer.