The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. Superoxide (O-2(center dot-)) is the proximal mitochondrial ROS, and in the present review 1 outline the principles that govern O(2)(center dot-)production within the matrix of mammalian mitochondria. The flux of O-2(center dot-) is related to the concentration of potential electron donors, the local concentration of O-2(center dot-) and the second-order rate constants for the reactions between them. Two modes of operation by isolated mitochondria result in significant O-2(center dot-) production, predominantly from complex I: (i) when the mitochondria are not making ATP and consequently have a high Delta p (protonmotive force) and a reduced CoQ (coenzyme Q) pool; and (ii) when there is it high NADH/NAD(+) ratio in the mitochondrial matrix. For mitochondria that are actively making ATP, and consequently have a lower Delta p and NADH/NAD(+) ratio, the extent of O-2(center dot-) production is far lower. The generation of O-2(center dot-) within the mitochondrial matrix depends critically on Delta p, the NADH/NAD(+) and CoQH(2)/CoQ ratios and the local O-2(center dot-) concentration, which are all highly variable and difficult to measure in vivo. Consequently, it is not possible to estimate O-2(center dot-) generation by mitochondria in vivo from O-2(center dot-) production rates by isolated mitochondria, and such extrapolations in the literature are misleading. Even so, the description outlined here facilitates the understanding of factors that favour mitochondrial ROS production. There is a clear need to develop better methods to Measure mitochondrial O-2(center dot-) and H2O2 formation in vivo,as uncertainty about these values hampers studies oil the role of mitochondrial ROS in pathological oxidative damage and redox signalling.