Granzyme B (GzmB) is a cytotoxic protease found in the granules of natural killer cells and cytotoxic T lymphocytes. GzmB cleaves multiple intracellular protein substrates, leading to caspase activation, DNA fragmentation, cytoskeletal instability, and rapid induction of target cell apoptosis. However, no known individual substrate is required for GzmB to induce apoptosis. GzmB is therefore thought to initiate multiple cell death pathways simultaneously to ensure the death of target cells. We previously identified Hop (Hsp70/Hsp90-organizing protein) as a GzmB substrate in a proteomic survey (Bredemeyer, A. J., Lewis, R. M., Malone, J. P., Davis, A. E., Gross, J., Townsend, R. R., and Ley, T. J. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 11785-11790). Hop is a co-chaperone for Hsp70 and Hsp90, which have been implicated in the negative regulation of apoptosis. We therefore hypothesized that Hop may have an anti-apoptotic function that is abolished upon cleavage, lowering the threshold for GzmB-induced apoptosis. Here, we show that Hop was cleaved directly by GzmB in vitro and in cells undergoing GzmB-induced apoptosis. Expression of the two cleavage fragments of Hop did not induce cell death. Although cleavage of Hop by GzmB destroyed Hop function in vitro, both cells overexpressing GzmB-resistant Hop and cells with a 90-95% reduction in Hop levels exhibited unaltered susceptibility to GzmB-induced death. We conclude that Hop per se does not set the threshold for susceptibility to GzmB-induced apoptosis. Although it is possible that Hop may be cleaved by GzmB as an "innocent bystander" during the induction of apoptosis, it may also act to facilitate apoptosis in concert with other GzmB substrates.