Expression of p60^c-src, the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60^c-src, was examined in mouse and rat authentic osteoclasts and mouse osteoclast-like multinucleated cells (MNCs) formed in vitro. In co-cultures of mouse osteoblastic cells and spleen cells, the expression of p60^c-src strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1α,25-dihydroxyvitamin D₃ (1α,25(OH)₂D₃). Immunohistochemical examination confirmed the high level expression of p60^c-src in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60^c-src was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60^c-src is important in osteoclastic bone resorption.