A tyrosine kinase, c-Src, that plays an indispensable role in ruffled border formation and bone resorption is constitutively active in osteoclasts. However, to date, the molecular mechanism underlying increased c-Src activity in osteoclasts is unknown. To address this, we first examined the expression levels and subcellular localization of Csk, a negative regulatory kinase for c-Src. We found that the expression level of Csk in osteoclasts was comparable with that of other tissues. However, in osteoclasts, Csk was hardly localized in lipid rafts, where c-Src is highly expressed. Interestingly, expression of Cbp, which recruits Csk into lipid rafts through physical interaction with Csk, was very low in osteoclasts compared with other tissues. To understand the importance of Cbp in osteoclasts, we introduced Cbp into osteoclasts using an adenovirus gene delivery system. Introduction of Cbp stimulated recruitment of Csk into lipid rafts and suppressed c-Src activity in a dose-dependent manner. Furthermore, introduction of Cbp markedly inhibited formation of actin rings and bone-resorbing activity in osteoclasts. In addition, treatment with RANKL and overexpression of TRAF6 or NFAT2 inhibited Cbp expression in the osteoclastogenic cell line RAW264.7 along with osteoclastic differentiation. NFAT2 overexpression also inhibited Cbp expression in spleen macrophages. Collectively, our results indicate that reduction in Cbp expression is responsible for maintaining high c-Src activity in osteoclasts. These findings contribute to an understanding of the unique regulatory system for c-Src in osteoclasts. (C) 2010 American Society for Bone and Mineral Research.