Cytotoxic T cells mobilize effector proteins from prestored lysosomal compartments. Since different activation signals result in alternative routes of target cell killing, utilizing either FasL or the granzyme B/perforin pathway, the existence of distinct forms of effector granules was recently suggested. Applying a protocol for the separation of intact organelles from activated T lymphoblasts, we noticed that FasL-associated secretory lysosomes (SL) segregate from vesicles containing larger amounts of granzymes and granulysin. We previously analyzed the proteome of secretory lysosomes from NK and T cells and now describe the proteome of granzyme-containing vesicles. Moreover, intact FasL-associated SL and granzyme-containing vesicles were compared by electron microscopy and respective extracts were characterized by Western blotting. With the present report, we provide a comprehensive proteome map of granzyme-containing granules and unequivocally demonstrate that T lymphoblasts contain at least two distinct types of effector vesicles. Moreover, the overall protein content of the two vesicle populations was compared by 2D difference gel electrophoresis. Interestingly, the observed differences in protein distribution were not restricted to effector proteins but also applied to cytoskeleton-associated elements that could argue for a differential transport or initiation of degranulation. To our knowledge, this is the first comprehensive description of distinct effector granules in T cells.