Abstract Interleukin-7 (IL-7) is a 25-kDa cytokine that was initially described as a pre-B cell growth factor and more recently has been shown to cause T cell proliferation. We have investigated the in vitro effects of IL-7 on mature T cells to include the generation and further expansion of allospecific and antitumor CTL. B6 anti-DBA allospecific CTL were generated in the presence of IL-7, IL-2, the combination IL-7 plus IL-2, or no cytokine. IL-7 alone or when combined with IL-2 enhanced the generation of allospecific CTL. To evaluate the proliferative effects of IL-7, 4-day B6 anti-DBA cultures were cultured in IL-7, IL-2, or no cytokine. Cell proliferation and duration of growth of cells cultured in IL-7 were significantly greater than cells cultured in IL-2 or in the absence of cytokine. Allospecific cytolytic activity was maintained during proliferation in IL-7 to a maximum of 60 days. In contrast with the ability of IL-2 to generate LAK cells, murine splenocytes cultured at varying doses of IL-7 (1 to 10,000 ng/ml), resulted in minimal LAK cell activity. The effect of IL-7 in the generation of CTL with antitumor activity was also studied. Seven days after footpad injection of MCA 203 or 205 sarcoma, draining lymph nodes (DLN) were harvested and restimulated in vitro with MCA 203 or 205, respectively, and maintained in culture with either IL-7, IL-2, the combination of IL-7 plus IL-2, or no cytokine. After 10 days in culture, cells generated in IL-7 or IL-2 exhibited similar cytotoxicity against the syngeneic autologous MCA tumor. IL-7 generated cells, however, showed specificity when tested by 51Cr release which was not seen with IL-2-generated cells. Cells generated in IL-7 plus IL-2 were more cytolytic than cells cultured with either cytokine alone. To further define the mechanism of action of IL-7 in antitumor CTL cultures, a monoclonal antibody, S4B6.1, capable of blocking murines-pecific IL-2 was employed. The partial inhibition by this mAb of the generation of antitumor CTL demonstrated that IL-7 acted, in part, by an IL-2-dependent mechanism. Finally, IL-7 cultures restimulated at Day 11 with autologous MCA 203 showed greater proliferation than IL-2 cultures and remained lytic at Day 21 of culture. We conclude that IL-7 is a potent T cell growth factor and may be useful for improving the generation of antitumor CTL for use in cellular immunotherapy.