BACKGROUND: Although increased levels of neutrophils and matrix metalloproteinase-9 (MMP-9) in the ischemic brain are reported in patients and experimental stroke studies the effects of neutrophils and MMP-9 on thromboembolic ischemic stroke are poorly understood. OBJECTIVES: To define the contribution of MMP-9 and neutrophils to brain injury. Blood-brain barrier breakdown (BBB) and hemorrhage after middle cerebral artery thromboembolism. METHODS: Thromboembolic stroke was induced in mice to determine the contribution of neutrophils and MMP-9 to acute stroke outcomes. The effects of MMP-9 gene deletion, specific MMP-9 inhibition, and antibody-mediated neutrophil depletion were examined. Ischemic brain injury, hemorrhage, swelling, neutrophil recruitment, MMP-9 expression, BBB breakdown, and fibrin(ogen) deposition were quantified. RESULTS AND CONCLUSIONS: During thromboembolic stroke, neutrophil recruitment and MMP-9 expression significantly increased in the ischemic brain hemisphere and were localized to the intravascular, perivascular, and brain parenchymal tissue. Neutrophil depletion significantly reduced brain swelling (P < .001) and cerebral infarction (P < .0001). Neutrophils colocalized with MMP-9, and nonneutrophilic expression of MMP-9 appeared vascular. MMP-9 deficiency significantly reduced neutrophil infiltration in the infarcted brain tissue (P < .01). MMP-9 deficiency (MMP-9-/- mice) and inhibition (anti-MMP-9 antibody) significantly decreased brain swelling (P < .001 and P < .05, respectively) and infarct volume (P < .05), but the effects were neutralized when the MMP-9-/- mice were supplemented with purified MMP-9 protein. MMP-9 deficiency/inhibition or neutrophil depletion caused comparable reductions in BBB breakdown and fibrin(ogen) deposition. However, even after stroke onset, intravenous treatment of MMP-9+/+ mice with an MMP-9-specific antibody significantly reduced brain infarction (P < .05). These data show that neutrophils and MMP-9 increase ischemic brain injury and BBB breakdown and that intravascular inhibition of MMP-9 may be a potential therapeutic strategy for ischemic stroke.