Recent reports have suggested that production of superoxide or other reactive oxygen species by activated osteoclasts may play a role in the complex process of bone resorption; however, the enzyme responsible for production of superoxide by osteoclasts has not been characterized. To determine if osteoclasts express NADPH-oxidase, a superoxide-generating enzyme found in phagocytic leukocytes, immunohistochemical studies were performed on tibia from 1-5-d-old rats using mAbs 449 and 48 and an antiserum specific for p47-phox. These antibodies recognize epitopes on the alpha and beta subunits of cytochrome b558, respectively, and the p47 cytosolic component of NADPH-oxidase. We found that osteoclasts attached to bone surfaces in tibia expressed all three components, as did mature polymorphonuclear and some mononuclear leukocytes in the bone marrow. In many adherent osteoclasts, the cytochrome b558 subunits were localized to the ruffled-border and bone interfaces. Studies were also performed on mature rat tibia that had undergone controlled fracture. By two weeks the healing fractures develop a callus rich in actively resorbing osteoclasts. Osteoclasts within the calluses, and attached to bone surface, also expressed the cytochrome b558 proteins. In addition to demonstrating the expression of NADPH-oxidase, the active production of superoxide by osteoclasts was also demonstrated in situ in freshly isolated tibia using 3,3'-diaminobenzidine (DAB)-Mn2+, a histochemical method specific for superoxide localization. Osteoclasts attached to bone surfaces contained deposits of oxidized DAB which were observed by light microscopy. Nonstimulated polymorphonuclear and mononuclear leukocytes in the bone marrow did not contain DAB deposits unless stimulated with phorbol myristate acetate, a known activator of NADPH-oxidase. These findings indicate that osteoclasts contain NADPH-oxidase, and during the process of resorbing bone, are actively producing superoxide.