Macrophage colony-stimulating factor (M-CSF) regulates the production, survival and function of macrophages through Fms, the receptor tyrosine kinase. Recently, interleukin-34 (IL-34), which shares no sequence homology with M-CSF, was identified as an alternative Fms ligand. Here, we provide the first evidence that these ligands indeed resemble but are not necessarily identical in biological activity and signal activation. In culture systems tested, IL-34 and M-CSF showed an equivalent ability to support cell growth or survival. However, they were different in the ability to induce the production of chemokines such as MCP-1 and eotaxin-2 in primary macrophages, the morphological change in TF-1-fms cells and the migration of J774A. 1 cells. Importantly, IL-34 induced a stronger but transient tyrosine phosphorylation of Fms and downstream molecules, and rapidly downregulated Fms. Even in the comparison of active domains, these ligands showed no sequence homology including the position of cysteines. Interestingly, an anti-Fms monoclonal antibody (Mab) blocked both IL-34-Fms and M-CSF-Fms binding, but another MAb blocked only M-CSF-Fms binding. These results suggested that IL-34 and M-CSF differed in their structure and Fms domains that they bound, which caused different bioactivities and signal activation kinetics/strength. Our findings indicate that macrophage phenotype and function are differentially regulated even at the level of the single receptor, Fms.