To clarify the mechanisms of bone destruction associated with bone metastases, we studied an animal model in which inoculation of MDA-MB-231 human breast cancer cells into the left cardiac ventricle of female nude mice causes osteolytic lesions in bone using morphological techniques. On the bone surfaces facing the metastatic tumor cells, there existed many tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. TRAP-positive mononuclear osteoclast precursor cells were also observed in the tumor nests. Immunohistochemical studies showed that the cancer cells produced parathyroid hormone-related protein (PTHrP) but not receptor activator of NF-kappaB ligand (RANKL). Histochemical and immunohistochemical examinations demonstrated that alkaline phosphatase and RANKL-positive stromal cells were frequently adjacent to TRAP-positive osteoclast-like cells. Immunoelectron microscopic observation revealed that osteoclast-like cells were in contact with RANKL-positive stromal cells. MDA-MB-231 cells and osteoclast-like cells in the tumor nests showed CD44-positive reactivity on their plasma membranes. Hyaluronan (HA) and osteopontin (OPN), the ligands for CD44, were occasionally colocalized with CD44. These results suggest that tumor-producing osteoclastogenic factors, including PTHrP, upregulate RANKL expression in bone marrow stromal cells, which in turn stimulates the differentiation and activation of osteoclasts, leading to the progression of bone destruction in the bone metastases of MDA-MB-231 cells. Because the interactions between CD44 and its ligands, HA and OPN, have been shown to upregulate osteoclast differentiation and function, in addition to the cell-cell interactions mediated by RANK and RANKL, the cell-matrix interactions mediated by these molecules may also contribute to the progression of osteoclastic bone destruction.