Estrogen-mediated neuroprotection is well established; however, no single mechanism of action for this effect has yet been established. As glial cells are integral for both the intact and injured nervous system, we hypothesized that estrogen-mediated neuroprotection may partly be attributed to attenuation of glial cell apoptosis, allowing them to protect neurons following injury. To assess the protective effects of estrogen on glia, C6 rat glioma cells were treated for 24 h with 500 microM glutamate. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and apoptosis was confirmed by cell morphology and DNA fragmentation. Pretreatment with 10 nM 17beta-estradiol (estrogen) increased cell viability and attenuated apoptosis. Treatment with the stereoisomer 17alpha-estradiol, or estrogen plus estrogen receptor antagonist ICI 182,780, was significantly less effective, indicating that cytoprotection was receptor-mediated. Estrogen treatment upregulated expression of estrogen receptor alpha. Cell impermeable bovine serum albumin-conjugated estrogen was also protective, indicating activation of estrogen receptors on the cell membrane. Intracellular free [Ca2+] was increased after glutamate treatment. This increase was attenuated in cells pretreated with estrogen. Glutamate increased the activity of pro-apoptotic proteases, such as calpain and caspase-3, and these protease activities were significantly attenuated by estrogen. The mechanism by which estrogen decreased intracellular Ca2+ was examined by assaying cell viability after using inhibitors that either blocked extracellular Ca2+ influx or prevented the release of intracellular Ca2+ stores. While several inhibitors increased cell viability in glutamate-treated cells, none were as protective as estrogen, and estrogen co-treatment significantly increased cell viability. These findings indicate that estrogen-mediated cytoprotection may be related to effects on Ca2+ entry but that these effects are not limited to any one of these Ca2+ entry points alone.