Osteoclast development requires cell-to-cell contact between hematopoietic osteoclast progenitors and bone marrow stromal/osteoblastic support cells. Based on this, we hypothesized that osteopontin, an adhesion protein produced by osteoclasts and osteoblasts, plays a role in osteoclastogenesis. Using in situ hybridization, we demonstrate that cells expressing the osteopontin messenger RNA (mRNA) appear after 3 days of culturing murine bone marrow cells. The number of these cells increases thereafter, reaching a peak on day 5. In the same cultures, cells expressing alkaline phosphatase (AP) or tartrate resistant acid phosphatase (TRAP), phenotypic markers for osteoblastic and osteoclast-like cells, respectively, appeared subsequent to the appearance of the osteopontin-positive cells. By means of a combination of in situ hybridization and histostaining, it was shown that the osteopontin mRNA was localized in 30–50% of the AP-positive or the TRAP-positive, as well as in nonspecific esterase (NSE)-positive, cells. The number of cells expressing both the osteopontin mRNA and either one of the three phenotypic markers was significantly increased in bone marrow cultures from estrogen-deficient mice, as compared with controls. Conversely, the number of all three populations of double positive cells was decreased in cultures treated with a specific antimouse rabbit osteopontin antibody or an RGD peptide. These findings indicate that osteopontin is expressed during the early stages of the differentiation of osteoclast and osteoblast progenitors in the bone marrow and that its cell adhesion properties are required for osteoclastogenesis.