The release of H2O2 from alveolar macrophages has been linked to the development of pulmonary fibrosis, but little is known about its source or mechanism of production. We found that alveolar macrophages from asbestosis patients spontaneously produce high levels of H2O 2 and have high expression of Cu,Zn-superoxide dismutase (SOD). Because Cu,Zn-SOD is found in the mitochondrial intermembrane space (IMS), we hypothesized that mitochondrial Cu,Zn-SOD-mediated H2O2 generation contributed to pulmonary fibrosis. Asbestos-induced translocation of Cu,Zn-SOD to the IMS was unique to macrophages and dependent on functional mitochondrial respiration and the presence of at least one of the conserved cysteines required for disulfide bond formation. These conserved cysteine residues were also necessary for enzyme activation and H2O 2 generation. Cu,Zn-SOD-mediated H2O2 generation was inhibited by knockdown of the iron-sulfur protein, Rieske, in complex III. The role of Cu,Zn-SOD was biologically relevant in that Cu,Zn-SOD-/- mice generated significantly less H2O 2 and had less oxidant stress in bronchoalveolar lavage fluid and lung parenchyma. Furthermore, Cu,Zn-SOD-/- mice did not develop pulmonary fibrosis, and knockdown of Cu,Zn-SOD in monocytes attenuated collagen I deposition by lung fibroblasts. Our findings demonstrate a novel mechanism for the pathogenesis of pulmonary fibrosis where the antioxidant enzyme Cu,Zn-SOD translocates to the mitochondrial IMS to increase H2O2 generation in alveolar macrophages. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.