Osteoclasts are derived from hematopoietic stem cells and their development is dependent on the products of stromal cells. CD9, a member of the tetraspan transmembrane-superfamily, is expressed on both hematopoietic cells and stromal cells. Addition of antagonistic rat anti-mouse CD9 antibody (KMC8.8) to cultures inhibited osteoclastogenesis on established stromal cell layers. When rat bone marrow cells depleted of adherent stromal cells were cultured on mouse stromal cells, numerous tartrate-resistant acid phosphatase-positive multinuclear cells were observed, and KMC8.8, which recognizes mouse but not rat CD9, completely prevented the generation of osteoclasts, suggesting that the CD9 expressed on the stromal cell is essential for osteoclastogenesis. Possibly for the same reason, KMC8.8 pretreatment of the mouse macrophage-like cell line C7, which is able to differentiate into mature osteoclasts, did not inhibit subsequent C7 cell differentiation, whereas the addition of KMC8.8 to cocultures of C7 cells with stromal cells inhibited the differentiation of C7 cells into osteoclasts. Moreover, we found that blockage of a signal via CD9 on stromal cells reduced transcription of the osteoclast differentiation factor (Odf) gene, which, together with macrophage colony-stimulating factor, is essential for osteoclastogenesis. These results revealed that CD9 molecules on stromal cells play a critical role in osteoclast development, possibly by modulating the expression of Odf.