ABSTRACT It has long been known that small changes to the structure of the R2 side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo, although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently, several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase, thereby preventing protein prenylation in osteoclasts. In this study, we examined the potency of a wider range of nitrogen- containing bisphosphonates, including the highly potent, heterocycle-containing zoledronic acid and minodronate (YM- 529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro, to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro, and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations $1 nM zoledronic acid or minodronate, the order of potency (zoledronic acid ' minodronate . risedronate . ibandronate . incadronate . alendronate . pamidronate) closely matching the order of antiresorptive potency. Furthermore, minor changes to the structure of the R2 side chain of heterocycle-containing bisphosphonates, giving rise to less potent inhibitors of bone resorption in vivo, also caused a reduction in potency up to ;300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo, and that small changes to the structure of the R2 side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase.