Background and Purpose— Perivascularly positioned cerebral mast cells (MC) have been shown to participate in acute blood-brain barrier disruption and expansive brain edema following experimental transient cerebral ischemia. However, the underlying molecular mechanisms remain unknown. Because proteolytic gelatinase enzymes, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to have a central role in compromising the integrity of the blood-brain barrier following ischemia, we examined whether cerebral MCs influence gelatinase activity in ischemic cerebral microvasculature. Methods— Rats underwent 60 minutes of middle cerebral artery occlusion followed by 3-hour reperfusion, and were treated with a MC-stabilizing (cromoglycate), or MC-degranulating (compound 48/80) agent, or vehicle. Genetically manipulated, MC-deficient WsRc ^Ws/Ws rats and their wild-type littermates (WT) underwent the same procedures. Cerebral edema and extravasation of Evans blue albumin were measured. Gelatinase activity was visualized by in situ zymography and was quantified with computerized high-throughput image and data analysis. Results— Activated MCs showed secretion of gelatinase-positive granules. Genetic MC deficiency decreased global gelatinase-active area (−69%, compared with WT; P <0.001) and the mean gelatinase activity of the ischemic microvasculature (−57% compared with WT; P =0.002). MC stabilization with cromoglycate decreased the percentage of microvessels with high gelatinase activity (−36% compared with saline; P <0.05). Compound 48/80 showed increased area of in situ zymography activity in the ischemic lesion (+55% compared with saline; P <0.001). Microvascular gelatinase activity correlated with brain swelling ( r =0.84; P <0.001; and r =0.61; P =0.02). Conclusions— Our data demonstrate that cerebral MCs participate in regulation of acute microvascular gelatinase activation and consequent blood-brain barrier disruption following transient cerebral ischemia.