A number of different organs and tissues from untreated or endotoxin-injected mice were surveyed for their ability to produce conditioned medium that could induce differentiation of the myelomonocytic leukemic cell line WEHI-3B. All organs were able to do this but were much more effective from endotoxin-injected animals. Production of the differentiation factor (DF) was dependent on protein synthesis and occurred throughout the 7-day incubation period. DF produced by the different organs in vitro and found in vivo in endotoxin serum were indistinguishable from each other by several fractionation procedures but could be distinguished from the majority of granulocyte-macrophage and macrophage colony-stimulating factors (GM- and M-CSFs). DF was precipitated by 55% ammonium sulfate whereas CSF required 85% saturation. DF did not bind to Concanavalin A-Sepharose whereas CSF did. DF eluted later than CSF from Phenyl-Sepharose columns. The dissociated molecular weight of DF was 21,000-29,000 and was not greatly affected by neuraminidase treatment. There was no evidence that DF was glycoprotein in nature although this cannot yet be ruled out. Although the DF activity could be clearly separated from the bulk of GM-CSF and M-CSF activities, there was always residual CSF activity associated with the DF. This CSF was distinct from GM- and M-CSF in its biochemical properties, in the fact that it only stimulated a subset of colony-forming cells and that upon dilution it stimulated exclusively granulocytic colonies to develop. In two steps (ammonium sulfate precipitation and Phenyl-Sepharose chromatography) it was possible to selectively enrich the differentiation activity over the CSF activity by 21-fold.