Abstract The present study was performed to clarify the role of vacuolar H +-ATPase in the regulation of the intracellular pH (pH i) in osteoclasts. Bafilomycin A 1or amiloride were added to rat osteoclast cultures to block the H +-ATPases and Na +/H +-exchangers, respectively. Addition of 10 −8M bafilomycin A 1to osteoclasts cultured on bone induced a rapid decrease of the pH i, while addition of amiloride had only a minor effect. The response to bafilomycin A 1appeared simultaneously with resorption activity and was abolished when resorption was inhibited by calcitonin. Osteoclasts on bone recovered from acid loading caused by propionate in the presence of amiloride, while bafilomycin A 1inhibited this recovery almost completely. The pH iof osteoclasts cultured on glass responded to the addition of amiloride, but was not effected by even high concentrations of bafilomycin A 1. In contrast, as little as 10 −10M bafilomycin A 1caused accumulation of large vesicles in the cytoplasm.