Osteoclasts are the main bone resorbing cells with capacity to acidify their intimate contact area with bone. Recent studies have suggested that osteoclast acid secretion is carried out by an H(+)-ATPase. We demonstrate here, that specific inhibitor of vacuolar type H(+)-ATPases, bafilomycin A1, inhibits bone resorption in osteoclast cultures as well as blocks proton transport in isolated medullary bone derived microsomes containing a vacuolar type H(+)-ATPase. These results demonstrate an important role of vacuolar H(+)-ATPase in bone resorption.