Cytotoxic T lymphocytes (CTL) kill virus-infected and tumorigenic target cells by releasing perforin from lytic granules (LG). CTL form a tight junction with the target cells called immunological synapse (IS). To avoid unwanted killing of neighboring cells, exocytosis of LG is tightly controlled and restricted to the IS. The mechanisms of LG accumulation and release at the IS is only partially understood. We show that in activated human primary CD8+ T cells, docking of LG at the IS requires tethering LG with CD3 containing endosomes (CD3-endo). Combining total internal reflection fluorescence microscopy and fast deconvolution microscopy (both in living cells) with confocal microscopy (in fixed cells), we found that LG and CD3-endo tether and are co-transported to the IS. Paired but not single LG are accumulated at the IS. The dwell time of LG at the IS is substantially enhanced by tethering with CD3-endo, resulting in a preferential release of paired LG over single LG. The SNARE protein Vti1b was found to be required for tethering of LG and CD3-endo. Down-regulation of Vti1b reduces tethering of LG with CD3-endo. This leads to an impaired accumulation, docking and release of LG at the IS. Therefore, Vti1b-dependent tethering of LG and CD3-endo determines accumulation, docking and efficient LG secretion at the IS. Our data and simulation of the data using a compartment model show that CTL need only a few minutes to secrete LG at the IS, supporting an en passant killing process of multiple targets simultaneously.