Abstract Ischemic neuronal injury mediated by cysteine proteases such as calpains and caspases has been demonstrated in various experimental models. Cathepsins B and L are also cysteine proteases which may contribute to neuronal death after ischemia. The authors measured in vitro and in vivo toxicity and post-ischemic cytoprotective effects of a cysteine protease inhibitor which does not block calpain or caspase but, rather, is relatively selective for cathepsins B and L. The compound belongs to the peptidyl-diazomethane family (cysteine protease inhibitor 1, termed CP-1). In vitro toxicity was measured using an assay of cell viability, and in vivo toxicity was measured by histological tissue analysis after infusion of CP-1 in rats. Two hours of middle cerebral artery (MCA) occlusion in rats was performed by the intravascular suture method. Immediately following reperfusion, intravenous infusion of CP-1 or vehicle was performed for 4 h at 0.9 ml/h. After a 7-day survival, the infarct volumes were measured. CP-1 was non-toxic to cultured glial cells to a local concentration of 200 μM, and relatively non-toxic to cultured endothelial cells at concentrations of 100–200 μM. No animal exhibited toxic effects at any of the doses used. Histologic comparisons revealed no signs of tissue toxicity. CP-1 significantly reduced hemispheric infarct volume compared to control (37±8.2%) at concentrations of 10, 50, and 250 μM [22±15%, P=0.008; 20±13%, P=0.002; 23±15%, P=0.022, respectively (mean±standard deviation; N=7–10 per group)]. CP-1, at the concentration of 50 μM, improved the functional score of the animals, but did not significantly alter cerebral blood flow. This study supports the hypothesis that the lysosomal cathepsins B and/or L contribute to cerebral injury after focal ischemia with reperfusion. Cysteine protease inhibitors which are relatively selective for cathepsins B and L, but not the calpains or caspases, are effective at reducing infarct volume after intravenous post-ischemic administration.