Estrogens are potent neuroprotective compounds in a variety of animal and cell culture models, and data indicate that estrogen receptor (ER)-mediated gene transcription is not required for some of these effects. To further address the requirement for an ER in estrogen enhancement of neuronal survival, we assessed the enantiomer of 17β-estradiol (Ent-E2), which has identical chemical properties but interacts only weakly with known ERs, for neuroprotective efficacy. Ent-E2 was both as potent and efficacious as 17β-estradiol in attenuating oxidative stress-induced death in HT-22 cells, a murine hippocampal cell line. Further, Ent-E2 completely attenuated H2O2 toxicity in human SK-N-SH neuroblastoma cells at a 10 nM concentration. In a rodent model of focal ischemia, 17β-estradiol (100 μg/kg) or Ent-E2 (100 μg/kg), injected 2 h before middle cerebral artery occlusion, resulted in a 60 and 61% reduction in lesion volume, respectively. Ent-E2, at the doses effective in this study, did not stimulate uterine growth or vaginal opening in juvenile female rats when administered daily for 3 days. These data indicate that the neuroprotective effects of estrogens, both in vitro and in vivo, can be disassociated from the peripheral estrogenic actions.