The requirements for generation of allospecific CTLs in vitro have been studied by "three cell" experiments, with two allogeneic T cell suspensions as cocultured responders, stimulated by irradiated B cells. This study describes enhancement as well as inhibition of the response, functionally defining T amplifier and T depressor cells regulating the differentiation of CTLs. Enhancement is the result of an amplifying effect of cytotoxic precursor T cells. Amplification is due to HLA-D region incompatibility between the responding T cell donor and the stimulating donor resulting in strong proliferation. Inhibition is the result of a depressing effect on cytotoxic precursor T cells mediated by cocultured T cells. The depression seems to be due to HLA-A, -B, -D identity between one of the responder T cell donors and the stimulator cells. The induction of depression is radiosensitive, accompanied by strong proliferation and CTL generation with cytotoxic specificity against the cocultured and depressed donor target cells. It is suggested that the functionally defined T depressor cells are cytotoxic precursors mediating cytostatic functions before strong cytotoxicity is detectable.