Reactive oxygen species (ROS) are thought to play a crucial role in determining longevity. ROS are produced by mitochondria and can attack and damage surrounding macromolecules. Accumulation of damage is suggested to be a major causal factor for the age-associated decline in physiological functions. There are several lines of supportive evidence, including observations that mitochondrial ROS production is lower in longer lived than shorter lived species and that rodents exposed to caloric restriction (which extends lifespan in a variety of organisms) have lower mitochondrial ROS production and lower macromolecular oxidative damage than controls. We studied the correlation between lifespan and mitochondrial ROS production in Drosophila melanogaster in two different models; in caloric restriction and in mutant flies over-expressing the mitochondrial adenine nucleotide translocase (ANT). Caloric restriction extended lifespan, but there was no difference in mitochondrial ROS production compared with control flies. Thus extension of lifespan is not necessarily linked to decreased mitochondrial ROS production. On the other hand, mitochondria from ANT over-expressers had significantly lower ROS production (because they had lower membrane potential), but the lifespan of flies over-expressing ANT was not extended compared to wild type. Thus decreased mitochondrial ROS production does not necessarily cause extension of lifespan. Our results show two examples in which mitochondrial ROS production and lifespan are not correlated, weakening the hypothesis that mitochondrial ROS production is an important determinant of lifespan.