Cytotoxic T and natural killer cells are able to kill their target cells through synergistic action of the pore-forming protein perforin and the serine protease granzyme B, resulting in very distinctive nuclear changes typical of apoptosis. Whereas perforin acts at the membrane, granzyme B appears to be both capable of entering the cytoplasm of target cells and accumulating in isolated nuclei. In this study we examine nuclear transport of fluoresceinated granzyme B both in vivo in intact cells in the presence of perforin and in vitro in semi-permeabilized cells using confocal laser scanning microscopy. Granzyme B alone was observed to enter the cytoplasm of intact cells but did not accumulate in nuclei. In the presence of sublytic concentrations of perforin, however, it accumulated strongly in intact cell nuclei to levels maximally about 1.5 times those in the cytoplasm after about 2.5 h. In vitro nuclear transport assays showed maximal levels of nuclear and nucleolar accumulation of granzyme B of about 2.5- and 3-fold those in the cytoplasm. In contrast to signal-dependent nuclear accumulation of SV40 large tumor antigen (T-Ag) fusion proteins in vitro, nuclear/nucleolar import of granzyme B was independent of ATP and not inhibitable by the non-hydrolyzable GTP analog GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)). Similar to T-Ag fusion proteins, however, granzyme B nuclear and nucleolar accumulation was dependent on exogenously added cytosol. Specific inhibitors of granzyme B protease activity had no effect on nuclear/nucleolar accumulation, implying that proteolytic activity was not essential for nuclear targeting. The results imply that granzyme B (32 kDa) may be transported from the cytoplasm to the nucleus through passive diffusion and accumulate by binding to nuclear/nucleolar factors in a cytosolic factor-mediated process. Active and passive nuclear transport properties were normal in the presence of unlabeled granzyme B, implying that the nuclear envelope and pore complex are not granzyme B substrates.