Estradiol has been shown to exert trophic and protective actions in the brain. Our laboratory has shown that in vivo, low physiological levels of estradiol protect the female rat brain against ischemic injury. In the present study, we used organotypic cortical explant cultures to begin to decipher the mechanisms of estradiol's actions. Injury was induced by exposure to kainic acid or potassium cyanide/2-deoxyglucose (KCN/2-DG) for varying lengths of time, and cell death was monitored by LDH release at 2, 6, 12, 24, 48, 72 and 96 h after injury. We found that exposure to 1 mM KCN/2 mM 2-DG for 2 h produced consistent delayed cell death that was detectable by 24 h. The presence of 17beta-estradiol (E2) during the 7 days prior to injury significantly reduced the extent of cell death; whereas, administration of E2 at the time of injury did not protect. The protective effects of estradiol were dose dependent. Low doses of E2 (1, 10, and 30 nM) significantly reduced cell death; however, higher concentrations of E2 (>60 nM) had no protective effect. The observations that low levels of E2 protect against cell death, and that pretreatment is required suggest that the protective actions of estradiol may involve estrogen receptors. Therefore, we examined the ability of 17alpha-estradiol, which does not efficiently activate the estrogen receptor, and the addition of the estrogen receptor antagonist, ICI 182,780, to influence the extent of cell death induced by KCN/2-DG. 17alpha-Estradiol failed to protect, and ICI 182,780 prevented E2 from protecting against cell death. Furthermore, E2 pretreatment is required for more than 24 h to be neuroprotective. Our results clearly show that in cortical explant cultures, estradiol protects cells against ischemic injury, and suggest that these protective actions involve estrogen receptors.