During the use of tetrapeptide and other proprietary caspase inhibitors in the study of neurodegeneration, we had concluded that mechanisms other than the inhibition of caspases contributed to the protective effects of certain caspase inhibitors. Here we report our studies to identify a target for and hence a mechanism by which the tetrapeptide inhibitor tyrosine-valine-alanine-aspartate-chloromethyl ketone (Ac-YVAD-cmk) is able to rescue neuronal cell cultures from cell death. Ac-YVAD-cmk rescued neuronal cells from cell death in response to oxidative stress and oxygen/glucose deprivation. Affinity labeling with biotinylated YVAD-cmk demonstrated distinct binding proteins for the inhibitor in cells from the central nervous system versus Jurkat cells. Binding to the novel target protein was displaced by class-specific protease inhibitors and suggested that the target is a cysteine protease. Affinity purification and sequencing identified the target as cathepsin-B. Cathepsin-B inhibitors competed with biotinylated YVAD-cmk for the target protein. The availability of the target for binding was reduced in cells that had been rescued by unlabeled inhibitor. Cathepsin-B inhibitors rescue hippocampal slices from cell death induced by oxygen/glucose deprivation. These data provide evidence to support a role for cathepsin-B in neuronal cell death, particularly that following ischemia.