Interleukin (IL)-3 has effects on a wide variety of cell types, including immature cells of the immune system, as well as mature cells such as granulocytes. We have investigated the effects of IL-3 on generation of cytolytic cells that can kill tumor cells. Previously, we have shown that IL-3 can enhance the development of cytolytic T cells (CTL) reactive with the line 1 tumor in a CD4-dependent manner. It is of interest that we found the development of CTL in response to IL-3 was not accompanied by increased development of nonspecific killer cells. This was in contrast to IL-2, which enhanced development of both CTL and nonspecific cells. To determine if IL-3 could inhibit the development of nonspecific killers, we added IL-3 to cultures of fresh spleen cells stimulated with high levels of IL-2. In this assay, IL-3 showed very potent inhibitory activity against the generation of nonspecific killers. To determine if IL-3 could inhibit the IL-2-driven generation of nonspecific killers in vivo, we injected a mixture of IL-2- and IL-3-producing line 1 cells. Each cytokine-producing transfectant was also injected alone. The cytotoxicity of tumor-infiltrating lymphocytes (TIL) from these tumors confirmed that the presence of IL-3 inhibits the generation of nonspecific killer cells in vivo.