Lymphokines induce the generation of cytotoxic cells (LICC) in the absence of antigenic or mitogenic stimulation. In the present study, we have demonstrated that at least two lymphokines are involved. They are interleukin 2-conditioned medium (CM-IL 2), which was produced by W/Fu rat spleen cells cultured with concanavalin A-conjugated Sepharose beads, and cytotoxic cell differentiation factor-conditioned medium (CM-CCDF), which was produced primarily by the unstimulated mouse peritoneal macrophages. It was first established that CCDF synergized with IL 2 to induce the generation of LICC in normal spleen cells, and that this process was specifically blocked by the rat anti-IL 2 receptor monoclonal antibody. The maximal synergistic effect was obtained by using 10% CM-CCDF (v/v) and 0.1 to 0.3 U/ml of CM-IL 2. Higher doses of IL 2 (3 to 10 U/ml) reduced the cytotoxic response. The effectors of LICC were Thy-1+, Lyt-2- and AGM1-; therefore, they were neither classic CTL nor NK cells. The precursors were AGM1+, Lyt-2- cells that were consistent of being NK-like cells. When examining the temporal relationship between CCDF and IL 2, we found that 4 hr preincubation of the responders with IL 2 was sufficient to activate the cytotoxic precursor cells. CCDF was needed later for the differentiation of the activated precursors into cytotoxic effectors. In contrast, preincubation of the responders with CCDF, followed by additional incubation with IL 2, failed to induce any cytotoxic response. These results established that lymphokine-induced cytotoxicity can be separated into two phases. In the activation phase, IL 2 provides the first signal to activate the cytotoxic precursors, with the process being completed in 4 hr. In the differentiation phase, CCDF provides the second signal to induce the differentiation of the IL 2-activated precursors into cytotoxic effectors, with this process requiring 48 hr to complete. The sequential presence of these lymphokines at an appropriate time during the activation and differentiation phases is critical for the generation of LICC response.