Human monocytes have been reported to release factors that can elicit distinct responses from a number of different target cells. Three of these activities are compared in this report. Supernatants of endotoxin-stimulated monocytes have been shown to stimulate the direct plaque response of T cell deficient splenocytes in vitro (BAF assay), to stimulate the thymidine incorporation by thymocytes (mitogen assay), and to synergize with phytohemagglutinin in the stimulation of thymocytes (PHA-enhancing assay). By gel filtration, plaque stimulating activity was recovered as a single peak with a m.w. of approximately 15,000 daltons when tested on nude splenocytes. When assayed on normal C57BL/10CR splenocytes, a second peak of plaque-stimulating activity was observed near 35,000 daltons. Mitogenic activity was observed both within the albumin peak and coincident with the 15,000 dalton plaque stimulator. Finally, PHA-enhancing activity was found to be broadly distributed between 8000 and 66,000 daltons, and included molecules that co-eluted with the 35,000 and 15,000 dalton plaque stimulators. Upon further purification of the 15,000 dalton material by isoelectric focusing, the plaque-enhancing, mitogenic, and PHA-enhancing activities co-eluted. This observation raised the possibility that all three activities of the 15,000 dalton material were due to the effects of a single factor on T cells. Some investigators favor this possibility based upon the study of the supernatants of human mixed leukocyte cultures (XRF). Here it is shown that spleen cell populations can be prepared that responded to BAF but not XRF, and vice versa. Therefore, studies on XRF cannot be used as evidence for the mechanism of the action of BAF. These results raise the possibility that this single factor may effect more than one target cell and may be responsible for a number of the sequellae of the stimulation of the reticuloendothelial system by bacterial products.