Osteoclasts resorb bone by transporting protons (H+) into the space between the cell and the bone. To investigate the roles of a sodium-hydrogen exchanger (NHE) and a vacuolar type H+-ATPase (V-ATPase) in H+ extrusion of osteoclasts attached to bone, changes in intracellular pH (pHi) were monitored in osteoclasts isolated on glass coverslips and on bone slices using a fluorescent pHi indicator. Acid-loaded osteoclasts on glass coverslips recovered their pHi in the presence of Na+. The pHi recovery was inhibited by 5-(N, N-hexamethylene) amiloride (HMA), an inhibitor of NHE, or by removal of Na+. 7-chloro-4-nitrobenz-2-oxa-1, 3-diazol (NBD-CI), a blocker of V-ATPase, did not block the H+ transport in osteoclasts on glass coverslips. Acid-loaded osteoclasts on bone slices recovered their pHi in the absence of Na+, and this recovery was blocked by NBD-Cl. In addition, neither HMA nor NBD-Cl inhibited the pH, recovery in the presence of Na+. These results indicated that osteoclasts attached to bone extrude H+ by both V-ATPase and NHE and that osteoclasts attached to glass mainly extrude H+ by NHE.