The high mobility group box-1 (HMGB1), originally identified as an architectural nuclear protein, exhibits an inflammatory cytokine- like activity in the extracellular space. Here we show that treatment with neutralizing anti- HMGB1 monoclonal antibody ( mAb; 200 mu g, twice) remarkably ameliorated brain infarction induced by 2- h occlusion of the middle cerebral artery in rats, even when the mAb was administered after the start of reperfusion. Consistent with the 90% reduction in infarct size, the accompanying neurological deficits in locomotor function were significantly improved. Anti- HMGB1 mAb inhibited the increased permeability of the blood- brain barrier, the activation of microglia, the expression of TNF-alpha and iNOS, and suppressed the activity of MMP- 9, whereas it had little effect on blood flow. Intracerebroventricular injection of HMGB1 increased the severity of infarction. Immunohistochemical study revealed that HMGB1 immunoreactivity in the cell nuclei decreased or disappeared in the affected areas, suggesting the release of HMGB1 into the extracellular space. These results indicate that HMGB1 plays a critical role in the development of brain infarction through the amplification of plural inflammatory responses in the ischemic region and could be an outstandingly suitable target for the treatment. Intravenous injection of neutralizing anti- HMGB1 mAb provides a novel therapeutic strategy for ischemic stroke.