The vitronectin receptor α_vβ₃ is highly expressed in osteoclasts and was shown to play a critical role in osteoclast function in vivo. The objective of this study was to examine the role of α_vβ₃ integrin in osteoclast formation in vitro using the inhibitory disintegrin echistatin, an RGD-containing snake venom. We documented by immunocytochemistry and Northern blot analysis that during murine osteoclast-like cell (OCL) formation in a coculture of mouse osteoblastic MB1.8 cells and bone marrow cells there is increased expression of the α_v and β₃ integrin subunits. Echistatin binds preferentially to the membrane fraction of isolated enriched OCLs (IC₅₀ = 0.6 nm), and this binding is inhibited by vitronectin receptor-blocking polyclonal antibodies. Additionally, cross-linking of radiolabeled echistatin to OCLs, followed by immunoprecipitation with antibodies to vitronectin or fibronectin receptors, shows that α_vβ₃ integrin is the predominant receptor for echistatin in this system. In this coculture, echistatin completely inhibits the formation of multinucleated OCLs, but not that of mononuclear prefusion OCLs (pOCs). This inhibition is RGD and dose dependent (IC₅₀ = 0.7 nm). We tested the hypothesis that inhibition of OCL formation may be due to interference with pOC migration and found that echistatin inhibited macrophage colony-stimulating factor-induced migration and fusion of pOCs (IC₅₀ = 1 and 0.6 nm, respectively). Echistatin inhibition of pOCs migration and fusion is also RGD dependent. These results suggest that the integrin α_vβ₃ plays a role in pOC migration, which can explain the inhibitory effect of echistatin on multinucleated osteoclast formation in vitro.