One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.