The proteinase responsible for bone collagen degradation in osteo-resorption was examined. The bone pit formation induced by parathyroid hormone (PTH) was markedly suppressed by leupeptin, E-64 and cystatin A, while no inhibition was observed by CA-074, a specific inhibitor of cathepsin B. Pig leucocyte cysteine proteinase inhibitor (PLCPI), a specific inhibitor of cathepsin L, and chymostatin, a selective inhibitor of cathepsin L, completely inhibited the pit formation. Cathepsin L activity in osteoclasts was much higher than the other cathepsin activities. Serum calcium in rats placed on a low calcium diet was decreased by treatment of E-64 or cystatin A, but not by CA-074. These findings suggest that cathepsin L is the main proteinase responsible for bone collagen degradation.