17beta-Estradiol (E(2)) was shown to exert neuroprotective effects both in in vitro and in vivo models of stroke. Although these effects of E(2) are known to require estrogen receptor-alpha (ER alpha), the cellular target of estrogen-mediated neuroprotection remains unknown. Using cell type-specific ER mutant mice in an in vivo model of stroke, we specifically investigated the role of ER alpha in neuronal cells versus its role in the microglia in the mediation of neuroprotection by estrogens. We generated and analyzed two different tissue-specific knockout mouse lines lacking ER alpha either in cells of myeloid lineage, including microglia, or in the neurons of the forebrain. Both E(2)-treated and E(2)-untreated mutant and control mice were subjected to a permanent middle cerebral artery occlusion for 48 h, and the infarct volume was quantified. Although the infarct volume of E(2)-treated female myeloid-specific ER alpha knockout mice was similar to that of E(2)-treated control mice, both male and female neuron-specific ER alpha mutant mice had larger infarcts than did control mice after E(2) treatment. We conclude that neuronal ER alpha in female and male mice mediates neuroprotective estrogen effects in an in vivo mouse model of stroke, whereas microglial ER alpha is dispensable.