Previous evidence has shown that Lyt-1+ helper T cells are required for an in vitro cytotoxic T cell response. We now present evidence which demonstrates an in vivo requirement for Lyt-1+ helper T cells in the activation of a killer T cell response. An adoptive transfer system was designed in which alloantigen-primed parental helper cells were transferred to an F1 recipient. After 1 day, this recipient of primed helper cells was lethally irradiated and normal responder spleen cells syngeneic to the primed helper cells were injected into the F1 recipient. In these experiments the semiallogeneic cells of the irradiated F1 mouse serve as the source of stimulator cells. When responder cells and irradiated helper cells were both present in the F1 mouse, good cytotoxic T cell responses were obtained. When either responders alone or helpers alone were examined, cytotoxic T cell responses were much lower. The presence in vivo of suppressor cells which inhibit cytotoxic T cell responses made it necessary to culture spleen cells from these irradiated recipients for short periods in vitro to reveal the cytotoxic T cells which had been generated in vivo. Cell collaboration was shown to occur before this in vitro culture period commenced; the generation of cytotoxicity required that helper cells and killer cell precursors interact in vivo. Treatment of the primed helper cells with anti-Thy-1 serum plus complement or anti-Lyt-1+ serum and complement removed helper cell activity. This indicates that the primed helper cell in in vivo collaboration is a Lyt-1+ T lymphocyte.