Estrogen provides neuroprotection against neurodegenerative diseases, including Parkinson's disease. Its effects may stem from interactions with neurons, astrocytes, and microglia. We demonstrate here in primary cultures of rat mesencephalic neurons that estrogen protects them from injury induced by conditioned medium obtained from lipopolysaccharide (LPS)-activated microglia. LPS-induced nitrite production and tumor necrosis factor-alpha up-regulation in microglia were blocked by estrogen pretreatment. Estrogen neuroprotection was related to microglial activation of estrogen receptors (ERs), insofar as the protective effect of the microglia-conditioned medium was overridden by pretreatment of microglia with the ER antagonist ICI 182,780. On the other hand, the specific ERalpha antagonist, MPP dihydrochloride, only partially blocked the effects of estrogen, suggesting that estrogen protection was mediated via both ERalpha and ERbeta. LPS treatment did not change ERalpha mRNA levels in microglia, astrocytes, and neurons, but it up-regulated ERbeta mRNA levels in microglia and astrocytes. Similarly, increased ERbeta protein levels were detected in LPS-activated microglia. More interesting was that immunocytochemical analysis revealed that ERbeta was localized in the cytoplasm of microglia and in the cell nucleus of astrocytes and neurons. In summary, our results support the notion that estrogen inhibits microglial activation and thus exhibits neuroprotective effects through both ERalpha and ERbeta activation. The cytoplasm location of microglial ERbeta suggests the possible involvement of nonclassical effects of estrogen on microglia. Changes in microglial ERbeta expression levels may modulate such effects of estrogen.