The present study uses the osteoclast precursor clonal line, HD-11EM, to study the potential of hydrogen peroxide (H 2 O 2 ) in mediating the differentiation of HD-11EM into osteoclast-like cells. HD-11EM cells are a newly established clonal cell line that, in response to 1a,25-(OH) 2 D 3 , differentiate into osteoclastlike cells that are multinucleated (more than three nuclei), express tartrateresistant acid phosphatase (TRAP), and excavate resorption pits when cultured on dentin slices in the presence of osteoblasts (Hsia et al., 1995, J. Bone Miner. Res., 10(Suppl 1):S424; Hsia, and Hauschka, 1997, unpublished data). Here we demonstrate that HD-11EM express the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase specific cytochrome b 558 subunits, and that stimulation of HD-11EM with 1 or 10 nM 1a,25-(OH) 2 D 3 increases the extracellular release of H 2 O 2 within 5 -10 min. Ours is the first report that stimulation of a cell with 1a,25-(OH) 2 D 3 enhances the activation of NADPHoxidase and increases the basal release of superoxide and the formation of its dismutation product, H 2 O 2 . To determine the possible involvement of H 2 O 2 in the differentiation of HD-11EM, these cells were exposed to glucose/glucose oxidase. This enzyme system was used to deliver a pure and continuous source of H 2 O 2 in nanomole amounts consistent with quantities produced by HD-11EM in response to 1a,25-(OH) 2 D 3 . Both 1a,25-(OH) 2 D 3 and the exogenously generated H 2 O 2 stimulated a dose-and time-dependent increase in TRAP activity/cell and the number of multinucleated cells 24-48 hr after treatment. Northern analysis confirmed an increase in expression of TRAP mRNA in response to either 1a,25-(OH) 2 D 3 or H 2 O 2 . Decreases in cell proliferation and v-myc mRNA were also observed in response to these agents. Taken together, our findings indicate that production of H 2 O 2 by HD-11EM is an important local factor involved in differentiation of HD-11EM into osteoclast-like cells, and suggest that H 2 O 2 may play a role in native osteoclast differentiation.