Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of NF-B ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption. Serum concentrations of RANKL are extremely high in OPG deficient (OPG^-/-) mice, suggesting that circulating RANKL is involved in osteoclastogenesis. RANKL^-/- mice exhibit osteopetrosis, with the absence of osteoclasts. We examined the requirements for osteoclastogenesis using OPG^-/- mice, RANKL^-/- mice and a system involving bone morphogenetic protein 2 (BMP-2)-induced ectopic bone formation. When collagen disks containing BMP-2 (BMP-2-disks) or vehicle were implanted into OPG^-/- mice, osteoclast-like cells (OCLs) and alkaline phosphatase-positive osteoblast-like cells appeared in BMP-2-disks but not in the control disks. F4/80-positive osteoclast precursors were similarly distributed in both BMP-2- and control disks. Cells expressing RANKL were detected in the BMP-2-disks, and the addition of OPG to the disk inhibited OCL formation. Muscle cells in culture differentiated into alkaline phosphatase-positive cells in the presence of BMP-2, and accordingly expressed RANKL mRNA in response to parathyroid hormone. This suggests that RANKL expressed by osteoblasts is a requirement for osteoclastogenesis. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL^-/- mice. BMP-2- and control disks were implanted into RANKL^-/- mice, which were injected with RANKL for 7 days. Many OCLs were observed in the BMP-2-disks and bone tissues but not in the control disks. These results suggest that osteoblasts also play important roles in osteoclastogenesis through offering the critical microenvironment for the action of RANKL.