Cytolytic granule-mediated target cell killing is effected in part through synergistic action of the membrane-acting protein perforin and serine proteases such as granzymes A (GrA) or B (GrB). In the present study we examine GrA cellular entry and nuclear uptake in intact mouse myeloid FDC-P1 cells exposed to perforin using confocal laser scanning microscopy, as well as reconstitute GrA nuclear uptake in vitro. GrA alone was found to be able to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of perforin, it specifically accumulated in the cell nuclei, with maximal levels about 2.5 times those in the cytoplasm after 2. 5 hours. In vitro, GrA accumulated in the nucleus and nucleolus maximally to levels that were four- and sixfold, respectively, those in the cytoplasm. In contrast, the active form of the apoptotic cysteine protease CPP32 did not accumulate in nuclei in vitro. Nuclear/nucleolar import of GrA in vitro was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPgammaS, but was dependent on exogenously added cytosol. Importantly, GrA was found to be able to accumulate in the nucleus of semi-intact cells in the presence of the nuclear envelope-permeabilizing detergent CHAPS, implying that the mechanism of nuclear accumulation was through binding to insoluble factors in the nucleus. GrB was found for the first time to be similar in this regard. The results support the contention that GrA and GrB accumulate in the nucleus through a novel nuclear import pathway, and that this is integral to induction of the nuclear changes associated with cytolytic granule-mediated apoptosis.