In the present study, we investigated changes of cytosolic Ca2+([Ca2+](cyt)), endoplasmic reticulum Ca2+([Ca2+](ER)) and mitochondrial Ca2+(Ca2+(m)) in astrocytes following oxygen/glucose deprivation and reoxygenation (OGD/REOX). Two hours OGD did not cause changes in [Ca2+](cyt), but led to a significant increase in [Ca2+](ER). The elevation in [Ca2+](ER) continued and reached a peak level (130 +/- 2 microM) by 90 min REOX. An abrupt release of Ca2+(ER) occurred during 1.5-2.5 h REOX, which was accompanied with a delayed and sustained rise in [Ca2+](cyt). Moreover, Ca2+(m) content was increased significantly within 15 min REOX followed by a secondary rise (approximately 4.5-fold) and a release of mitochondrial cytochrome c (Cyt c). Astrocytes exhibited translocation of Cyt c from mitochondria to endoplasmic reticulum (ER) and up regulation of ER stress protein p-eIF2alpha. Blocking Na+-K+-Cl(-) cotransporter isoform 1 activity, either by its potent inhibitor bumetanide or genetic ablation, abolished release of ER Ca2+, delayed rise in [Ca2+](cyt) and Ca2+(m). Inhibition of the reverse mode operation of the Na+/Ca2+ exchanger significantly attenuated OGD/REOX-mediated Cyt c release. In summary, this study illustrates that OGD/REOX triggers a time-dependent loss of Ca2+ homeostasis in cytosol and organelles (ER and mitochondria) in astrocytes. Collective stimulation of Na+-K+-Cl(-) cotransporter isoform 1 and reverse mode function of Na+/Ca2+ exchanger contributes to these changes.