Spleen cells from mice treated with cyclophosphamide (100 to 200 mg/kg) were unable to generate effective cytotoxic thymus-derived cells to allogeneic tumor cells in vitro. The inability of lymphoid cells from cyclophosphamide-treated mice to generate thymus-derived cytotoxic cells became more apparent as the number of responding cells became limited. This depressed response was not due to the elimination of cytotoxic precursor cells since normal response levels were restored by the addition of thymus cells. The added thymus cells did not provide cytotoxic cells in the culture. The thymus cells active in restoring cytotoxic activity were sensitive to mitomycin C, cyclophosphamide, and anti-theta serum and complement. In addition, the active thymus cells were located in the cortisone-resistant pool and did not adhere to nylon wool columns.