Tissue plasminogen activator (tPA) induces hemorrhagic transformation (HT) after stroke and the incidence of hyperglycemia further exacerbates this injury. Matrix metalloprotease 3 (MMP3) amplifies the tPA-induced HT. The influence of the interaction between tPA and HG on activating MMP3 after stroke was not previously reported. Accordingly, we investigated the impact of tPA and HG on MMP3 activity and the mechanisms through which they induce MMP3 activation in hyperglycemic stroke. Methods. Control/normoglycemic and hyperglycemic (blood glucose: 140-200 mg/dl) male Wistar rats were subjected to either middle cerebral artery (MCA) suture occlusion for 90 minutes or thromboembolic occlusion and up to 24 h reperfusion, with or without tPA (1 mg/kg, IV, 2 h after stroke). MMP3 activity and MMP3 tyrosine nitration were evaluated in brain homogenates at 24 h. Brain vascular endothelial cells (BVEC) were subjected to 3 h hypoxia under either normal or high glucose conditions with or without tPA. MMP3 activity and MMP3 tyrosine nitration were assessed at 24 h. Results. HG and tPA significantly increased MMP3 activity in the brain after stroke and in BVECs after hypoxia/reoxygenation. HG significantly increased MMP3 tyrosine nitration in rats subjected to either suture or thromboembolic occlusion as well as in BVECs (Table). Conclusion. HG and tPA significantly increased MMP3 activity in the brain after stroke and this was associated with increased MMP3 nitration. Our findings suggest that tyrosine nitration may be the underlying mechanism through which MMP3 is activated in hyperglycemic stroke.