The study investigates the mechanism of action of the bisphosphonate alendronate in rat bone. [³H]-alendronate was used to label bone particles and its localization in rat bone was examined 1 and 6 days after injection. Alendronate bound to bone particles with a Kd of ~1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5, binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 × 10−3 fmol/μm2, which would produce a concentration of 0.1–1 mM in the resorption space if 50% were released. At these concentrations, membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, and interferes with bone resorption and ruffled border formation without destroying the osteoclasts.The study investigates the mechanism of action of the bisphosphonate alendronate in rat bone. [³H]-alendronate was used to label bone particles and its localization in rat bone was examined 1 and 6 days after injection. Alendronate bound to bone particles with a Kd of ~1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5, binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 × 10−3 fmol/μm2, which would produce a concentration of 0.1–1 mM in the resorption space if 50% were released. At these concentrations, membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, and interferes with bone resorption and ruffled border formation without destroying the osteoclasts.