Killer lymphocytes utilize the synergy of a membranolytic protein, perforin, and the serine protease granzyme B (grB) to induce target cell apoptosis, however the mechanism of this synergy remains incompletely defined. We have previously shown that perforin specifically induces the redistribution of cytoplasmic grB into the nucleus of dying cells, however a causal role for nuclear targeting of grB in cell death has not been demonstrated. In the present study, we used confocal laser scanning microscopy (CLSM) to determine whether the nuclear accumulation of fluoresceinated (FITC-) grB precedes or is a consequence of apoptosis. Two distinct and mutually exclusive cellular responses were observed in FDC-P1 cells: (i) up to 50% of the cells rapidly accumulated FITC-grB in the nucleus (maximal at 7 min; t1/2 of 2 min) and underwent apoptosis; (ii) the remaining cells took up FITC-grB only into the cytoplasm, and escaped apoptosis. Under these conditions, DNA fragmentation was not observed for at least 13 min, indicating nuclear accumulation of grB preceded the execution phase of apoptosis. Furthermore, nuclear import of grB proceeded through an intact nuclear membrane, as the nuclei of cells whose cytoplasm was pre-loaded with 70 kDa FITC-dextran excluded dextran for up to 90 min while still undergoing apoptosis in response to perforin and grB. These findings indicated that perforin-induced nuclear accumulation of grB precedes apoptosis, and is not a by-product of caspase-induced nuclear membrane degradation. The cell membrane lesions formed by perforin in these experiments were not large enough to permit a 13 kDa protein (yeast cdk p13suc) access into the cytoplasm, but an 8 kDa protein (bacterial azurin) was able to equilibrate between the cytosol and the exterior. Therefore, transmembrane pores large enough to allow passive diffusion of grB (32 kDa) into the cell are not necessary for apoptosis. Rather, a perforin-dependent signal results in a redistribution of grB from the cytoplasm to the nucleus, where it may contribute to the nuclear changes associated with apoptosis.