During focal cerebral ischemia, the degradation of microvessel basal lamina matrix occurs acutely and is associated with edema formation and microhemorrhage. These events have been attributed to matrix metalloproteinases (MMPs). However, both known protease generation and ligand specificities suggest other participants. Using cerebral tissues from a non-human primate focal ischemia model and primary murine brain endothelial cells, astrocytes, and microglia in culture, the effects of active cathepsin L have been defined. Within 2 hours of ischemia onset cathepsin L, but not cathepsin B, activity appears in the ischemic core, around microvessels, within regions of neuron injury and cathepsin L expression. In in vitro studies, cathepsin L activity is generated during experimental ischemia in microglia, but not astrocytes or endothelial cells. In the acidic ischemic core, cathepsin L release is significantly increased with time. A novel ex vivo assay showed that cathepsin L released from microglia during ischemia degrades microvessel matrix, and interacts with MMP activity. Hence, the loss of microvessel matrix during ischemia is explained by microglial cathepsin L release in the acidic core during injury evolution. The roles of cathepsin L and its interactions with specific MMP activities during ischemia are relevant to strategies to reduce microvessel injury and hemorrhage.