The dynamics of lytic granule contents during CTL differentiation and following CTL activation have previously not been fully characterized. Murine T cell activation leads to an extensive architectural reorganization of the lysosomal compartment with the transition from few LAMP1+ small cytoplasmic vesicles to numerous large LAMP1+ vesicular structures. Human CTL clone activation leads to the progressive association of GrA to LAMP1-2+ organelles, suggesting that lytic molecules accumulate within pre-existing or newly formed lysosomes. Rapidly after an effector CTL encounters a target cell presenting cognate antigenic peptides, lytic granules are delivered into the intercellular cleft, which correlates with the appearance of LAMP1 at the CTL surface and the concurrent loss of intracellular lytic molecules. It is considered that lytic granules are not intrinsically prone to secretion, since their exocytosis NSC 136476 requires the acquisition of molecules promoting membrane docking and fusion, such as Rab27a and Munc14-3, via the fusion with endosomal/ exocytic vesicles. Most studies on lytic granules are based on confocal and electron microscopy. Therefore a quantitative assessment of lytic granule composition and load during their biogenesis and maturation is still missing. Here, we developed a flow cytometry approach to characterize the composition of individual lytic granules deriving from primary human CTL. Our study indicates that during CTL differentiation, lytic granules arise from the stepwise loading of lytic molecules into lysosomes. Our approach furthermore reveals that, following activation, differentiated CTL recruit Rab27a+ only on a set of lytic granules and that they do not secrete all of them. Despite the biological significance of lytic granules as key organelles delivered by CTL and NK cells to lyse infected or malignant host cells, the composition of the lytic granule population during its biogenesis and secretion has previously only been partly characterized. Here, we report a novel approach based on flow cytometry to study the expression levels of membraneassociated molecules and intra-granular lytic molecules in large numbers of lytic granules. This approach was applied to primary human CTL at different stages of differentiation and activation, to precisely measure load in lytic molecules and association with the docking molecule Rab27a. Out of the crude vesicular extract of CTL, lytic granules were identified based on the presence of both membrane-associated and intra-granular molecules. Differentiated CTL contained a relatively homogeneous pool of lytic granules co-expressing all the above-mentioned molecules. This apparent homogeneity is somewhat in contrast with the analysis of EM pictures showing that lytic granules harbor heterogeneous morphological appearances, as previously reported in murine T cells and human clones. The analysis of lytic granule diameter size distribution by flow cytometry assisted by size standard beads also failed to discriminate lytic granule subsets since most lytic granules distributed in a broad but continuous fashion from a few hundreds of a mm to a few mm, as previously noted.