The osteoclasts, which play an essential role in bone resorption, are multinucleated cells (MNCs). They are formed by the fusion of mononuclear preosteoclasts (pOCs). However, because of the difficulty of isolating the pOCs, the process of fusion of pOCs has not been elucidated. To establish a fusion assay system, we succeeded in isolating pOCs that are not contaminated with MNCs and osteoblastic cells from the coculture of mouse bone marrow cells and osteoblastic cells. When pOCs were cultured in the presence of osteoblastic cells, the fusion of pOCs into MNCs took place within 24 h. No MNCs were formed in the absence of osteoblastic cells. The number of MNCs formed by the fusion of pOCs was dependent on the number of both pOCs and osteoblastic cells in the culture. Osteoblastic cells were also required for bone resorption by the osteoclasts that were formed by the fusion of pOCs. Osteotropic hormones, such as 1α,25-dihydroxy vitamin D3 [1α,25(OH)2D3] and parathyroid hormone (PTH), did not affect the fusion and pit formation of pOCs in the absence of osteoblastic cells. Eel calcitonin (eCT), however, significantly inhibited the fusion of pOCs induced by osteoblastic cells. These results suggest that the fusion of pOCs was induced by the direct contact between pOCs and osteoblastic cells..