The excretion of cathepsin B, a lysosomal cysteine proteinase, by parathyroid hormone-stimulated embryonic mouse calvaria in culture, correlates closely with the extent of bone resorption evaluated by the loss of hydroxyproline and calcium and by the extension of resorption lacunae. E-64, a specific inhibitor of cysteine proteinases, inhibits reversibly the resorption of cultured bones without affecting the hormone-induced secretion of lysosomal hydrolases. Given in vivo to rats, the proteinase inhibitors, E-64 and leupeptin, both induce a concomitant fall in the serum calcium level and in the urinary excretion of hydroxyproline. These results provide evidence that cysteine proteinases, possibly lysosomal cathepsins, are necessary for bone resorption.