To investigate the hypothesis that caloric restriction alters mitochondrial function in situ, intact hepatocytes were isolated from fully fed and calorie-restricted (55% of control food intake, 4 months duration) male Brown-Norway rats at 6 months of age, and various parameters were determined. Overall, the production of reactive oxygen species was not affected by caloric restriction, neither were the mitochondrial membrane potential, oxygen consumption driving proton leak, or oxygen consumption driving ATP turnover. It is concluded that while isolated mitochondria from liver tissue of calorie-restricted animals display a reduction in the generation of reactive oxygen species, it was not possible to confirm this effect in isolated hepatocytes. Further work is required to establish what effect, if any, caloric restriction has on the rate of generation of reactive oxygen species in intact cells and tissues and importantly at the whole-animal level.