A variety of approaches, including the use of a monoclonal antibody, have indicated that the lymphokine Interleukin 2 is a necessary and sufficient mediator for initiating cytotoxic T cell differentiation in vitro. Mitogen (Con A, PHA) stimulation of spleen cells and K/D or I region stimulation in primary MLC all led to IL-2 production, as measured by the growth of an IL-2 dependent T cell clone, and to the production of a "helper" factor necessary for cytotoxic T lymphocyte (CTL) differentiation against metabolically inactivated stimulator spleen cells. Several lines of evidence suggested that the IL-2 activity and CTL "helper" activity were due to a single molecular entity. These included the observations that: i) There was a linear relationship (R = 0.99) between IL-2 activity and relative CTL "helper" activity regardless of the preparation used (purified IL-2; MLC, or mitogen-induced supernatants). ii) Absorption of MLC supernatants with T cell "blasts" or with IL-2 dependent T cell lines concomitantly removed both IL-2 and CTL "helper" activities. iii) IL-2 activity purified by isoelectric focusing, or by polyacrylamide gel electrophoresis, displayed CTL "helper" activity. iv) Both CTL "helper" and IL-2 activities were neutralized by a monoclonal antibody directed against IL-2.