Four of 12 monoclonal antibodies (MoAbs) directed to different epitopes in the extracellular domain of the human colony-stimulating factor-1 receptor (CSF-1R, the c-fms proto-oncogene product) specifically inhibit CSF-1 binding to receptor-bearing cells. All four antibodies abrogated CSF-1-dependent colony formation by human bone marrow-derived macrophage precursors and by mouse NIH-3T3 cells expressing a transduced human c-fms gene. In addition, one of these antibodies (designated MoAb 2–4A5) interfered with the ligand-independent proliferation of NIH-3T3 cells transformed by an oncogenic, mutant c- fms allele. Unlike CSF-1 itself, neither MoAb 2–4A5 nor the other three inhibitory antibodies (MoAbs 12–2D6, 12–3A1, and 12–3A3) induced CSF-1R internalization or degradation. These antibodies should prove useful not only for identifying and quantitating CSF-1R on receptor-bearing cells but for abrogating specific receptor signals that govern the proliferation and survival of human mononuclear phagocytes.