The neuroprotective effects of estrogen were studied in the ischemic model mice by 90 min transient unilateral middle cerebral artery occlusion (MCAO) followed by 22.5 h reperfusion. The total infarct size in C57BL/6 female mice after MCAO and reperfusion was significantly smaller than that in male mice. Intraperitoneal injection of estrogen after the start of reperfusion significantly reduced the infarct volume in the male mice. However, no significant gender difference was found in total infarct size in gamma protein kinase C (PKC)-knockout mice, suggesting that the neuroprotective effects of estrogen are due to the activation of a specific subtype of PKC, gammaPKC, a neuron-specific PKC subtype, in the brain. We demonstrated that exogenous estrogen-induced neuroprotection was attenuated in gammaPKC-knockout mice. Immunocytochemical study showed that gammaPKC was translocated to nerve fiber-like structures when observed shortly after MCAO and reperfusion. We also visualized the rapid and reversible translocation of gammaPKC-GFP (green fluorescent protein) by estrogen stimulation in living CHO-K1 cells. These results suggest that the activation of gammaPKC through the G-protein-coupled estrogen receptors on the plasma membrane is involved in the estrogen-induced neuroprotection against focal brain ischemia.