Integrins that bind RGD (arginine-glycine-aspartic acid) containing peptides, especially the vitronectin receptor alpha(v)beta3, have been implicated in the regulation of osteoclast function. Echistatin, an RGD-containing snake venom peptide with high affinity for beta3 integrins, as well as nonpeptide RGD mimetics, were shown to inhibit osteoclastic bone resorption in vitro and in vivo. To evaluate the role of RGD-binding integrins in bone metabolism, we examined by several methods the effects of echistatin on ovariectomy (OVX)-induced bone loss in mice and rats. First, we confirmed that echistatin binds in vitro with high affinity (Kd, 0.5 nM) to alpha(v)beta3 integrin purified from human placenta and established a competitive binding assay to measure echistatin concentrations in serum. We find that echistatin infused for 2 or 4 weeks at 0.36 microg/h x g body weight (approximately 50 nmol/day x mouse) completely prevents OVX-induced cancellous bone loss in the distal femora of ovariectomized mice. Echistatin has no effect on uterine weight, body weight, and femoral length changes induced by OVX, nor does it cause any apparent changes in major organs other than bone. In OVX rats, echistatin infusion at 0.26 microg/h x g for 4 weeks effectively prevents bone loss, evaluated by dual energy x-ray absorptiometry of the femur, by femoral ash weight, and by bone histomorphometry of the proximal tibia. At effective serum concentrations of 20-30 nM, measured at the end of the infusion period, echistatin maintains histomorphometric indices of bone turnover at control levels but does not decrease osteoclast surface. In conclusion, these results provide in vivo evidence, at the level of bone histology, that RGD-binding integrins, probably alpha(v)beta3, play a rate-limiting role in osteoclastic bone resorption and suggest a therapeutic potential for integrin ligands in the suppression of bone loss.