We have established a method for obtaining an enriched preparation of functionally active osteoclast-like multinucleated cells (enriched OCLs) from co-cultures of mouse primary osteoblasts and bone marrow cells. Using these enriched OCLs, the effect of osteoblastic cells on osteoclast function was examined in two assays: a pit formation assay and an assay for actin ring formation. The enriched OCLs cultured for 24 h on dentine slices formed only a few resorption pits. When various numbers of primary osteoblasts were added to the enriched OCLs, the areas of the resorption pits increased proportionally to the number of osteoblasts added. Like primary osteoblasts, the established cell lines of osteoblastic cells (MC3T3-E1 and KS-4) and bone marrow-derived stromal cells (MC3T3-G2/PA6 and ST2) potentiated the pit formation caused by enriched OCLs. In contrast, the fibroblastic cell lines NIH3T3 and C3H10T1/2) and the myoblastic cell line (C2C12) failed to activate OCL function. When cell-to-cell contact between MC3T3-E1 cells and enriched OCLs was prevented, only a few resorption pits were formed. Pit formation by enriched rat osteoclasts placed on dentine slices was also stimulated by adding MC3T3-E1 cells. Actin ring formation and pit forming activity were well correlated in either culture of enriched mouse OCLs or authentic rat osteoclasts on dentine slices. These results indicate that osteoclast function is activated by osteoblastic cells through a mechanism involving cell-to-cell and/or cell-to matrix contact.