After our previous report that osteoclast-like multinucleated cells (MNCs) were formed in response to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] in cocultures of mouse spleen cells and osteoblast-rich populations freshly isolated from fetal mouse calvariae, we examined whether such primary osteoblast-like cells can be replaced by established cell lines in inducing osteoclast-like cell formation. We first used two clonal cell lines simultaneously established from newborn mouse calvariae. One was the osteoblastic cell line MC3T3-E1, and the other was the preadipose cell line MC3T3-G2/PA6. Tartrate-resistant acid phosphatase (TRACP; a marker enzyme of osteoclasts)-positive mononuclear cells and MNCs were formed in the cocultures of spleen cells and MC3T3-G2/PA6 cells in the presence of 1 alpha,25-(OH)2D3. Dexamethasone greatly potentiated TRACP-positive MNC formation induced by 1 alpha,25-(OH)2D3, whereas the glucocorticoid alone had no effect on it. In contrast, osteoblastic MC3T3-E1 cells failed to induce TRACP-positive cells in the cocultures. Another bone marrow-derived stromal cell line ST2 also induced TRACP-positive MNC formation in the cocultures in response to 1 alpha,25-(OH)2D3 and dexamethasone. Salmon calcitonin enhanced cAMP production in the cocultures only when TRACP-positive cells were formed. Autoradiographic studies demonstrated that [125I]calcitonin specifically bound to TRACP-positive cells formed in the cocultures. When spleen cells and either MC3T3-G2/PA6 or ST2 cells were cocultured on sperm whale dentine slices in the presence of 1 alpha,25-(OH)2D3 and dexamethasone, numerous resorption lacunae were formed. These results show that the two bone marrow-derived stromal cell lines can support osteoclast-like cell differentiation in cocultures with spleen cells.