AbstractSpleen cells from BALB/c mice immunized with mitomycin C-treated MOPC 104E plasmacytoma cells demonstrated negligible cytotoxic activity (<10% specific cytotoxic activity) in the51Cr release assay. These cells exhibited increased cytotoxic activity when they were secondarily sensitized in vitro with mitomycin C-treated MOPC 104E cells. Spleen cells from normal mice showed tumor-specific cytotoxic activity when cocultured with mitomycin C-treated tumor cells at the optimal responder to stimulator ratio of 25:1. The level of cytotoxic activity obtained by in vivo primed and secondarily in vitro sensitized spleen cells did not exceed the level of activity obtained by in vitro primary sensitized cells. Significant suppression of the cytotoxic activity of in vitro primary sensitized cells was observed when cocultured with in vivo primed spleen cells during primary sensitization in vitro at a responder to suppressor cell ratio of 1:1. Suppressor cells of in vivo primed mice were removed by treatment with anti-Thy 1.2 and complement. These results suggest that spleen cells from in vivo primed mice consisted of at least two subpopulations of cells, a cytotoxic (prekiller) and suppressor T cells. Attempts to induce cytotoxic cells in vivo might have failed because of the appearance of suppressor T cells.