Cultures of peritoneal exudate cells rich in macrophages were studied for the secretion of lymphostimulatory molecules. Two conditions produced increased secretion: (a) addition to the cultures of various agents that readily interacted with macrophages, such as latex particles, antibody-coated red cells, endotoxin, Listeria organisms, or Be salt or (b) addition of activated lymphocytes. In the first case the increased activity was found during the first 24 or 48 h after uptake of the stimuli. Increased activity was found in normal or peptonestimulated macrophages but not in macrophages after injection of endotoxin or thioglycollate. The addition of T lymphocytes from Listeria-infected mice to macrophage cultures increased greatly the activities. This increase was also produced by addition of antigen-primed T cells together with antigen. The lymphocytes by themselves did not secrete active factors. The lymphostimulatory activities were tested on thymocyte DNA synthesis and on antibody formation in vitro. The latter assay was done on spleen cells from immunized mice where one striking effect was the stimulation of differentiation to antibody-secreting cells. Some dissociation of both activities (thymocyte DNA synthesis and B-cell differentiation) was observed with selected culture fluids.