Rac small GTPases may play an important regulatory role in osteoclastogenesis. Our in vitro and in vivo results show that both Rac1 and Rac2 are required for optimal osteoclast differentiation, but Rac1 is more critical. Rac1 is the key Rac isoform responsible for regulating ROS generation and the actin cytoskeleton during the multiple stages of osteoclast differentiation.Introduction: Recent evidence suggests that the Rac small GTPases may play an important regulatory role in osteoclastogenesis. This finding is important because bisphosphonates may regulate their antiresorptive/ antiosteoclast effects through the modification of Rho family of small GTPases. Materials and Methods: To elucidate the specific roles of the Rac1 and Rac2 isoforms during osteoclastogenesis, we used mice deficient in Rac1, Rac2, or both Rac1 and Rac2 in monocyte/osteoclast precursors. Macrophage-colony stimulating factor (M-CSF)-and RANKL-mediated osteoclastogenesis in vitro was studied by using bone marrow-derived mononucleated preosteoclast precursors (MOPs). The expression of osteoclast-specific markers was examined using quantitative real-time PCR and Western blot analysis. Free actin barbed ends in bone marrow MOPs after M-CSF stimulation was determined. The ability of MOPs to migrate toward M-CSF was assayed using Boyden chambers. Margin spreading on heparin sulfate-coated glass and RANKL-induced reactive oxygen species generation were also performed. Functional assays of in vitrogenerated osteoclasts were ascertained using dentine sections from narwal tusks. Osteoclast levels in vivo were counted in TRACP and immunohistochemically stained distal tibial sections. In vivo microarchitexture of lumbar vertebrate was examined using CT 3D imaging and analysis. Results: We show here that, although both Rac isoforms are required for normal osteoclast differentiation, Rac1 deletion results in a more profound reduction in osteoclast formation in vitro because of its regulatory role in pre-osteoclast M-CSF-mediated chemotaxis and actin assembly and RANKL-mediated reactive oxygen species generation. This Rac1 cellular defect also manifests at the tissue level with increased trabecular bone volume and trabeculae number compared with wildtype and Rac2-null mice. This unique mouse model has shown for the first time that Rac1 and Rac2 play different and nonoverlapping roles during osteoclastogenesis and will be useful for identifying the key roles played by these two proteins during the multiple stages of osteoclast differentiation. Conclusions: Rac1 and Rac2 play different and nonoverlapping roles during osteoclastogenesis. This model showed that Rac1 is the key Rac isoform responsible for regulating ROS generation and the actin cytoskeleton during the multiple stages of osteoclast differentiation.