Estrogens and androgens influence many properties of breast cancer cells and, hence, regulation of local estrogen and androgen levels by enzymes involved in steroid hormone biosynthesis and metabolism would impact signaling by these hormones in breast cancer cells. In this study we show that the UDP-glucuronosyltransferase enzyme UGT2B15, a member of the UGT family of Phase II enzymes involved in the glucuronidation of steroids and xenobiotics, is a novel, estrogen-regulated gene in ER-positive human breast cancer cells (MCF-7, BT474, T47D and ZR-75). UGT2B15 is the only UGT2B enzyme up-regulated by estrogen, and marked estradiol stimulation of UGT2B15 mRNA levels is observed, in a time- and dose-dependent manner. UGT2B15 stimulation by estradiol is blocked by the antiestrogen ICI 182,780, but not by the translational inhibitor cycloheximide, indicating that UGT2B15 is likely a primary transcriptional response mediated through the estrogen receptor. UGT2B15 up-regulation is also evoked by other estrogens (propyl-pyrazole-triol, genistein) and by the androgen 5-DHT working through the estrogen receptor, but not by other steroid hormone receptor ligands. Western blot and immunocytochemical analyses with several UGT2B-specific antibodies we have designed, and steroid glucuronidation assays, indicate a large increase in both cellular UGT2B15 protein and enzyme activity following estrogen treatment. Due to the important role of UGT enzymes in forming conjugates between steroids and glucuronic acid, thereby inactivating them and targeting them for removal, the estrogen-induced up-regulation of UGT2B15 might have a significant moderating effect on estrogen and androgen concentrations, thereby reducing their signaling in breast cancer cells.