Macrophage colony-stimulating factor (MCSF) and osteoprotegerin ligand (OPGL), both produced by osteoblasts/stromal cells, are essential factors for osteoclastogenesis. Whether local MCSF levels regulate the amount of osteoclast formation is unclear. Two culture systems, ST-2 and Chinese hamster ovary-membrane-bound MCSF (CHO-mMCSF)-Tet-OFF cells, were used to study the role of mMCSF in osteoclast formation. Cells from bone marrow (BMM) or spleen were cultured with soluble OPGL on glutaraldehyde-fixed cell layers; osteoclasts formed after 7 days. Osteoclast number was proportional to the amount of soluble OPGL added. In contrast, varying mMCSF levels in the ST-2 or CHO-mMCSF-Tet-OFF cell layers, respectively by variable plating or by addition of doxycycline, did not affect BMM osteoclastogenesis: 20-450 U of mMCSF per well generated similar osteoclast numbers. In contrast, spleen cells were resistant to mMCSF: osteoclastogenesis required 250 U per well and further increased as mMCSF rose higher. Our results demonstrate that osteoclast formation in the local bone environment is dominated by OPGL. Increasing mMCSF above basal levels does not further enhance osteoclast formation from BMMs, indicating that mMCSF does not play a dominant regulatory role in the bone marrow.