Both human and murine osteoclasts can be derived in vitro from hematopoietic cells or monocytes that are cocultured with osteoblasts or marrow-derived stromal cells. The osteoclastogenic stimulus provided by murine osteoblasts and marrow-derived stromal cells is now known to be mediated by osteoclast differentiation factor (ODF), a membrane-bound tumor necrosis factor-related ligand. This study demonstrates that mouse spleen cells and monocytes form osteoclasts when cultured in the presence of macrophage-colony stimulating factor (M-CSF) and a soluble form of murine ODF (sODF). Numerous multinucleated osteoclasts expressing tartrate resistant acid phosphatase (TRAP) and calcitonin receptor (CTR) formed within 7 days of culture and engaged in extensive lacunar bone resorption. Osteoclast number and bone resorption area was dependent on sODF concentration. Long-term cultured human monocytes also formed bone resorbing osteoclasts in response to co-stimulation by sODF and M-CSF, although this required more than 11 days in culture. This human osteoclast differentiation was strongly inhibited by granulocyte-macrophage colony stimulating factor. This study further characterises murine osteoclast differentiation caused by sODF and M-CSF co-stimulation in vitro, and shows that the same co-stimulation causes human osteoclast differentiation to occur. We propose that this methodology can be employed to investigate the direct effects of cytokines and other factors on human osteoclast differentiation.