Reactive oxygen species (ROS), formed during normal aerobic metabolism, are involved in signal transduction and cognitive functions, but highly increased ROS concentrations may also have detrimental effects. The aim of the present study was to investigate whether aging and cognitive functions are associated with ROS generation in human neocortex obtained from neurosurgical patients. ROS formation in mitochondria from fresh and re-thawed neocortical specimens was measured by monitoring ROS-mediated conversion of dihydrorhodamine 123 to fluorescent rhodamine 123. The validity of this technique was characterized in rat brain mitochondria. The increase in the concentration–response curve of the complex I inhibitor rotenone on ROS generation, as measured by rhodamine 123 (Rh123) fluorescence, was much more pronounced than that of rotenone on mitochondrial [3H]-choline uptake [which indicates changes in the mitochondrial membrane potential (ΔΨ M)]. Thus, mitochondrial ROS generation can be shown by Rh123 fluorescence although this fluorescence may also reflect changes in ΔΨ M to some extent. ROS formation in human brain mitochondria positively correlated with the age of patients. Moreover, an age-corrected positive correlation of ROS formation with presurgical cognitive performance was observed. Our data suggest a mild increase in ROS formation with aging possibly reflecting a physiological compensation of mitochondrial function. Furthermore, higher cognitive performances in tests of executive functions may be paralleled by slightly increased ROS levels.