The role of the inducible matrix metalloproteinase (MMP)-9 in blood–brain barrier (BBB) disruption after ischemic stroke is well accepted. Recombinant tissue plasminogen activator (r-tPA) is the only approved thrombolytic treatment of ischemic stroke but r-tPA is potentially neurotoxic. Vasogenic edema after r-tPA treatment has been linked with an increase in cerebral MMP-9. However, because cerebral ischemia clearly increases the levels of endogenous tPA, the consequence of additional r-tPA may be questionable. In this study, wild type and MMP-9 knockout mice were subjected to 90 min transient middle cerebral artery occlusion and treated with 10 mg/kg r-tPA. At 24 h after occlusion, BBB permeability, hemispheric enlargement, collagen and laminin degradation as well as cerebral infarction were increased in both wild type and MMP-9 knockout treated animals as compared with non-treated animals. Mortality was increased in wild type but reduced in knockout treated mice. Cerebral MMP-9 concentration was not modified by r-tPA. However, pre-treatment with p-aminobenzoyl-gly-pro-D-leu-D-ala-hydroxamate, a broad-spectrum MMP inhibitor, counteracted the effects of r-tPA on the neurovascular unit and decreased mortality in both wild type and knockout mice. MMP inhibition did not modify cerebral infarction in r-tPA-treated animals. Our results suggest that r-tPA toxicity is mainly independent of MMP-9 after transient middle cerebral artery occlusion but could involve some other MMPs. Additionally, our results support the hypothesis of a dissociation between r-tPA-dependent mechanisms of BBB breakdown and cerebral infarction. Due to the importance of r-tPA in thrombolytic treatment of ischemic stroke patients, the MMPs that could participate in r-tPA-induced BBB disruption should be further characterized.