Supernatants from phorbol myristate acetate (PMA)-stimulated EL4.IL2 cells (EL4.PMA), but not recombinant IL-2 (rIL-2), induced the production of cytotoxic T lymphocytes (CTL) in low density murine spleen cell cultures. CTL induction in these cultures was completely abrogated by treatment with anti-Thy-1 or anti-Lyt-2 antibody plus complement but not by anti-L3T4 antibody plus complement. Fractionation of EL4.PMA on a Sephadex G-150 column demonstrated that the CTL-inducing activity in EL4.PMA eluted with an apparent molecular weight of about 44,000 and was partially separated from IL-2. This 44,000 MW material was shown to contain insignificant amounts of PMA. Following a 3-day culture period with the partially purified factor, C57BL/6J thymocytes could proliferate and differentiate into cytotoxic cells in response to rIL-2, whereas there was no proliferation or generation of cytotoxic cells when the thymocytes were cultured in rIL-2 alone. The number of IL-2 receptor-positive cells in C57BL/6J thymocytes also increased from 1.1% to 22.8% after 3 days of culture in the partially purified factor. Recombinant IL-4 (BSF-1) and IL-5 (TRF), when used alone or in combination with rIL-2, were unable to induce a cytotoxic response under similar culture conditions. These findings are consistent with the interpretation that EL4.PMA contains a novel lymphokine that directly, or indirectly, induces the expression of IL-2 receptors on resting CTL precursors without intentional stimulation by specific antigen. In the presence of IL-2, these precursors may then differentiate into effector CTL.