Mitogen-induced cytotoxicity was studied by culturing mouse spleen cells with an optimum mitogenic dose of concanavalin A (Con A) for 2 days and, after washing, assessing their ability to lyse tumor targets. We show that rapid lysis occurs only when the correct concentration of an agglutinating mitogen (phytohemagglutinin P (PHA) or Con A) is present in the assay. PHA is more efficient than Con A in revealing the cytotoxic effect. The Con A-induced effector cytotoxic cell is shown to be sensitive to anti-ϑ serum and complement. Cytotoxic T cells were also induced by H-2 different allo-immunization. These effector cells specifically lyse targets which bear the immunizing H-2 antigens in the absence of PHA. When PHA is present in the assay, they will also lyse syngeneic tumor targets nonspecifically. Since not all dividing T cells (e.g., T lymphomas, and T blasts induced by M locus different, H-2 similar mixed lymphocyte culture) lyse PHA-P815, we propose that this assay measures only a subset of effector T cells, namely, cytotoxic T cells, regardless of their antigen specificity. The tumor cells, P815 and EL4, are sensitive both to antigen-specific T cell-mediated lysis in the absence of PHA and to nonspecific, PHA-revealed lysis. Small lymphocytes, T cell blasts, and B cell blasts are sensitive to lysis by T cells which are directed against H-2 antigens on their surface, but are not very susceptible to nonspecific T cell lysis in the presence of PHA. The reason for this difference in susceptibility of tumor and normal cells to PHA-revealed nonspecific T cell lysis is not known but may have some relevance to in vivo tumor rejection.