Drug-mediated tumor antigens (DMTA), which appear after in vivo treatment of murine lymphomas with 5-(3,3'-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC), are shown to elicit strong primary cell-mediated responses in vitro. Specific cytotoxic response is obtained using CD2F1 (H-2d/H-2d) spleen cells cocultured in vitro for 5 days with inactivated histocompatible L5178Y/DTIC lymphoma cells in a one-way mixed lymphocyte tumor culture. Sensitized lymphoid cells are markedly cytotoxic for L5178Y/DTIC targets and are minimally or not active against the parental lymphoma. Marginal or no cross-reactivity is found between L5178Y/DTIC lymphoma and other untreated or DTIC-treated tumor or normal cells. Cytotoxic activity is T dependent, since it is abrogated by treatment in vitro with anti-θ serum and complement and is not elicited by spleen cells of athymic (nude) donors.