Osteoclasts dissolve bone through acidification of an extracellular compartment by means of a multimeric vacuolar type H+-ATPase (V-ATPase). In mammals, there are four isoforms of the 100-kDa V-ATPase "a" subunit. Mutations in the a3 isoform result in deficient bone resorption and osteopetrosis, suggesting that a3 has a unique function in osteoclasts. It is thus surprising that several studies show a basal level of a3 expression in most tissues. To address this issue, we have compared a3 expression in bone with expression in other tissues. RNA blots revealed that the a3 isoform was expressed highest in bone and confirmed its expression (in decreasing order) in liver, kidney, brain, lung, spleen, and muscle. In situ hybridization on bone tissue sections revealed that the a3 isoform was highly expressed in multinucleated osteoclasts but not in mononuclear stromal cells, whereas the a1 isoform was expressed in both cell types at about the same level. We also found that a3 expression was greater in osteoclasts with 10 or more nuclei as compared with osteoclasts with five or fewer nuclei. We hypothesize that these differences in a3 expression may be associated with previously demonstrated differences between large and small osteoclasts with reference to their resorptive activity.