Cytotoxic lymphocytes induce target cell apoptosis via two major pathways: Fas/FasL and granule exocytosis. The latter pathway has largely been defined by the roles of the pore-forming protein perforin and by the serine proteinases granzymes A and B. Upon entry into target cells, the granzymes cleave substrates that ultimately result in cell death. To gain further insight into granzyme B function, we have identified novel substrates. SDS-PAGE analysis of S100 cell lysates identified a 51 kDa protein that was cleaved by granzyme B. Mass spectrometry analysis revealed that this fragment was the microtubule protein, alpha-tubulin, which was confirmed by western blotting. In addition, two-dimensional gel analysis showed that the truncated form of alpha-tubulin had a more basic isoelectric point than the full-length molecule, suggesting that granzyme B removed the acidic C-terminus. Site-directed mutagenesis within this region of alpha-tubulin revealed the granzyme B recognition site, which is conserved in a subset of alpha-tubulin isoforms. Significantly, we showed that alpha-tubulin was cleaved in target cells undergoing apoptosis as induced by cytotoxic T lymphocytes. Therefore, in addition to its role in the activation of mitochondria during apoptosis, these results suggest a role for granzyme B in the dismantling of the cytoskeleton.