Our preliminary studies showed 17 ?-estradiol (E2)-regulated Ca2+ homeostasis may play a role in enhancing nitric oxide (NO) production in endothelial cells. Here, we investigated whether the effect of E2 on intracellular calcium concentration ([Ca2+]i) regulation in human endothelial cells (EA.hy926) was estrogen receptor (ER) dependent. Using PCR, we showed ERα but not ER? was expressed in EA.hy926. To establish the role of ERα in E2-regulated Ca2+ homeostasis, ERα was down-regulated in EA.hy926 using siRNA (90 pmol) as determined by real time RT-PCR and Western blotting. [Ca2+]i in Fura 2 loaded ERα knockdown cells and negative control knockdown cells was measured using spectrofluorometry. In the absence of extracellular Ca2+, inhibition of endoplasmic reticulum Ca2+-ATPase with thapsigargin (1 ?M) caused a single Ca2+ transient response, indicating Ca2+ release from the store. Cells subjected to siRNA-mediated ERα knockdown treated with E2 (1 ?M, 24 h) exhibited reduced TG-induced Ca2+ release compared to E2 treated negative control knockdown cells. Furthermore, a subsequent increase in extracellular Ca2+ concentration led to a robust increase in [Ca2+]i which was significantly reduced in E2 treated ERα knockdown compared to E2 treated control cells. Our results indicated that ERα may contribute to E2-regulated Ca2+ homeostasis and subsequently NO production in endothelial cells. Supported by NHLBI.