According to the state of mitochondrial respiration, the respiratory chain generates superoxide anions converted into hydrogen peroxide. Two uncoupling proteins (UCP) able to modulate the coupling between the respiratory chain and ATP synthesis are now identified and could be involved in mitochondrial H₂O₂ generation. UCP1 is specific to brown adipose tissue (BAT) whereas UCP2 is expressed in numerous tissues, particularly in monocytes/macrophages. Preincubation of BAT mitochondrial fractions with GDP, an inhibitor of UCP1, induced a rise in mitochondrial membrane potential (assessed by rhodamine 123 uptake) and H₂O₂ production. An uncoupling agent reversed this effect. Liver mitochondria exhibited a similar phenotype. GDP was also able to raise membrane potential and H₂O₂ production of the mitochondria from nonparenchymal cells expressing UCP2, but was completely ineffective on mitochondria from hepatocytes deprived of UCP2. The GDP effect was also observed with mitochondrial fractions of the spleen or thymus, which highly expressed UCP2. Altogether, these results strongly suggest that UCP2 is sensitive to GDP and that the UCPs, particularly UCP2, are able to modulate H₂O₂ mitochondrial generation. This supports a role for UCP2 in cellular (patho‐) physiological processes involving free radicals generated by mitochondria, such as oxidative damage, inflammation, or apoptosis.—Nègre‐Salvayre, A., Hirtz, C., Carrera, G., Cazenave, R., Troly, M., Salvayre, R., Pénicaud, L., Casteilla, L. A role for uncoupling protein‐2 as a regulator of mitochondrial hydrogen peroxide generation. FASEB J. 11, 809–815 (1997)