The localization of vacuolar H+-ATPase (V-ATPase) in osteoclasts of rat tibiae was examined immunohistochemically using antibodies to 57kD and 72 kD subunits of V-ATPase, with confocal laser scanning microscopy and transmission electron microscopy. The two antibodies showed similar immunoreactivities in osteoclasts. We distinguished three patterns of immunoreactivity in osteoclasts: The first pattern was that immunoreactivity was diffusely detected in cytoplasm of osteoclasts; The second one was that osteoclasts showed intense immunoreactivity in their regions contacting with bone surface; and the last was that little immunoreactivity was seen in osteoclasts. Post-embedding methods with protein A-gold complex revealed that the subunits of V-ATPase were localized not only on the membranes of the ruffled borders of osteoclasts, but also around the Golgi apparatus and accumulated tubular lysosomes.