One mechanism cytotoxic T lymphocytes use to kill targets is exocytosis of cytotoxic agents from lytic granules, a process that requires Ca 2 + influx. We investigated the role of Ca 2 + influx in granule exocytosis using TALL-104 human leukemic cytotoxic T cells triggered via a bispecific antibody containing an anti-CD3 F(ab') to kill Raji B lymphoma cells. Using a novel fluorescence method, we detected target-directed release of ~15% of lytic granules during killing. Consistent with previous work, we observed sustained CTL Ca 2 + gradients during killing, but gradients reflect the behavior of Fura-2 in granules. Rapid imaging experiments suggest that Ca 2 + channels are not polarized during killing, indicating that Ca 2 + influx does not direct granule reorientation. Furthermore, we find that Ca 2 + acts via a high-affinity interaction to promote granule exocytosis.