The article discusses the sites and topology of mitochondrial superoxide production, which is an important source of reactive oxygen species (ROS) in cells and may contribute to aging and age-related diseases. Seven major sites of superoxide production in mammalian mitochondria are identified, including the ubiquinone binding sites in complex I and complex III, glycerol 3-phosphate dehydrogenase, the flavin in complex I, ETFQOR, and pyruvate and 2-oxoglutarate dehydrogenases. The topology of these sites is crucial because it determines whether superoxide produced in the mitochondrial matrix can damage mitochondrial DNA. All sites produce superoxide in the matrix, and some also produce superoxide in the intermembrane space. The relative contribution of each site to mitochondrial ROS generation under various conditions is not fully understood, and it varies with species, tissue, substrate, energy demand, and oxygen tension. The article also reviews the mitochondrial free radical theory of aging, the mechanisms of ROS production, and the challenges in measuring ROS production in isolated mitochondria and cells.The article discusses the sites and topology of mitochondrial superoxide production, which is an important source of reactive oxygen species (ROS) in cells and may contribute to aging and age-related diseases. Seven major sites of superoxide production in mammalian mitochondria are identified, including the ubiquinone binding sites in complex I and complex III, glycerol 3-phosphate dehydrogenase, the flavin in complex I, ETFQOR, and pyruvate and 2-oxoglutarate dehydrogenases. The topology of these sites is crucial because it determines whether superoxide produced in the mitochondrial matrix can damage mitochondrial DNA. All sites produce superoxide in the matrix, and some also produce superoxide in the intermembrane space. The relative contribution of each site to mitochondrial ROS generation under various conditions is not fully understood, and it varies with species, tissue, substrate, energy demand, and oxygen tension. The article also reviews the mitochondrial free radical theory of aging, the mechanisms of ROS production, and the challenges in measuring ROS production in isolated mitochondria and cells.