Cytolytic T lymphocytes (CTL) were generated in secondary mixed leukocyte-tumor cell cultures (MLTC) with syngeneic RB1-5 tumor cells as stimulating cells and with responding spleen cells from regressor mice that had rejected a murine sarcoma virus (MSV)-induced tumor. CTL precursor cells were found to be exclusively of thymic origin and non-T cells were apparently not required for CTL generation. When the size variations of CTL from syngeneic MLTC were analyzed by velocity sedimentation it appeared that a transition from small precursor cells to larger effector cells occurred during the first 5 days in culture; this change in cell size was then followed by a shift toward small-sized cells. Furthermore, the CTL generated in syngeneic MLTC in the MSV tumor immune system were compared with those CTL obtained in allogeneic mixed leukocyte cultures (MLC) and were shown to exhibit fundamental similarities.