In this study we show that T cells from mice infected with Listeria monocytogenes can interact in vitro with normal macrophages to produce a number of soluble mediators, including lymphostimulatory molecules. One of these molecules was a 15,000-dalton protein mitogenic for thymocytes. Generation of mitogenic activity was essentially completed by the first 24 hr of culture and did not require the addition of Listeria antigens. Production of mitogenic protein required contact between the lymphocytes and macrophages, because it did not occur when the two cells were separated by a cell-impermeable membrane. Optimal production of mitogenic protein occurred only when the lymphocytes and macrophages shared homologous I-A regions of the major histocompatibility complex. Once generated, the mitogenic protein did not display histocompatibility restriction and could stimulate allogeneic as well as syngeneic thymocytes. Strains of mice with the C57 background responded poorly to mitogenic protein even though those strains were capable of producing it. We conclude that an early stage in T cell immunity to Listeria involves an intimate association with macrophages regulated by the H-2 complex.