Culture fluids of peritoneal exudate cells rich in macrophages stimulated DNA synthesis of thymocytes and, to lesser extent, of spleen cells. The authors investigated the effects of culture fluids from macrophages on the in vitro response to a hapten carrier protein (fluorescein hemocyanin) using spleen cells from immune mice. Macrophage culture fluids contained an activity that increased the plaque forming cell response of both IgG and IgM class. This increase was observed in the absence of any added hapten protein to the culture. The helper function of T lymphocytes (as evidenced by challenging with the hapten on the homologous carrier) was also increased by the macrophage culture fluid. However, this enhancement was best observed in conditions of relatively low T cell activity. Also, the macrophage fluid allowed spleen cells of nude athymic mice to make a plaque forming cell response to sheep red blood cells of both the IgM and IgG class. The macrophage was the cell source of the stimulatory molecule since it was generated only in cultures of macrophages devoid of significant number of lymphocytes. Stimulatory activity was not found in cultures of lymphocytes, mouse embryo cells, or 3T3 cells. The thymocyte stimulatory molecule did not contain H 2 antigens, was resistant to diisopropylfluorophosphate treatment, eluted from Sephadex with a size ranging from 15,000 to 21,000 daltons, and was sensitive to chymotrypsin and pepsin.