Cytoplasmic granules from cytotoxic T lymphocytes and other lymphoid cells were purified by the Percoll gradient technique previously shown to yield pure cyto-plasmic granules from cytotoxic LGL tumors. Such granules purified from cloned CTL lines were shown to be comparable cytolytic activity (on a per cell basis) to the LGL tumore granules. Cytolytic activity is associated with lysosomal enzymes in the Percoll gradient, and is seen on a variety of nucleated cells as well as on red cells. The lytic process is rapid, occurring within 15 minutes at room temperature, and is absolutely dependent on calcium in the medium. Addition of calcium to purified cloned CTL granules give rise to membrane-associated ring structures with internal diametes of 5-10 nM as seen in the EM. These are smaller than those produced by the LGL cytolysin, and there are other subtle differences in the lytic activity. CTL granules caused carboxyfluorescein release from liposomes in a rapid and calcium-dependent process. Granules prepared from primary CTL cultures gave a calcium dependent cytolytic activity, but this was about 10-50 fold less than the cloned CTL on a per cell basis. Normal lymphocytes from spleen, thymus and the T cells from peripheral blood gave granules with no cytolytic activity, as did most lymphoid tumor cells. The mouse T cell tumor BW5147 gave a weakly cytolytic granule preparation. Two cloned CTL lines having no cytolytic activity gave granules of comparable cytolytic capacity to the CTL lines, but oher cloned T lymphocytes gave non-cytolytic granules. CTL granule-mediated lytic activity was specifically neurtralized by rabbit antibodies raised against the LGL tumor granules, but these antibodies did not inhibit CTL activity.