Subcellular particulate membrane fragments prepared from murine lymphoid or tumor cells were used as a cell-free antigen source in order to stimulate the generation of cytolytic thymus-derived effector cells (CTL) in vitro. When cultivated with normal spleen cells as a source of responding lymphocytes, particulate antigen preparations induced only low CTL activities; in contrast, virtually normal responses were observed when immune spleens were used as a source of responding cells. In both cases, results were compared to those responses obtained with intact irradiated (1000 rads) normal spleen cells as stimulating antigen. The effector cells generated with particulate antigen preparations (obtained either by hypotonic shock or by sonication) were shown to be T cells and were characterized with regard to the kinetics of their response and their dose-activity relationship. It was also shown that the responses observed were specific, both at the level of initiation and at the effector level. The results obtained suggest that there exists a fundamental difference between normal and alloimmune spleen cells in their ability to respond in vitro to stimulation by subcellular antigen preparations.