Cell-mediated cytotoxic responses in vitro to surface antigens associated with murine sarcoma virus (MSV)-induced tumors were investigated using mixed leukocyte-tumor cell cultures (MLTC). The source of responding cells was either spleens from normal C57BL/6 mice (primary MLTC) or spleens of C57BL/6 mice carrying or having rejected a MSV-induced tumor (secondary MLTC). Graffi virus-induced GiL-4 leukemia cells, Rauscher virus-induced RB1-5 leukemia cells, and MSV-induced MSV-B16 sarcoma cells were used as stimulating syngeneic tumor cells and/or target cells. Under appropriate culture conditions, cytolytic T lymphocytes (CTL) were generated in both primary and secondary MLTC. As assessed by a quantitative short-term 51Cr release assay system, CTL activity in secondary MLTC populations was at least 10-fold higher than that in primary MLTC populations, and 100-fold higher than that in spleen cells taken at the peak of the in vivo response of MSV-infected mice. The ability of spleen cells to mount a secondary CTL response in vitro could be observed as early as 5 days after virus injection, increased up to the time of maximum tumor size and persisted long after tumor regression. This suggests the development of increased numbers of CTL progenitors and/or the formation of "memory" CTL in spleens of MSV-injected mice.