Calorie restriction (CR) has been shown to decrease H(2)O(2) production in liver mitochondria, although it is not known if this is due to uniform changes in all mitochondria or changes in particular mitochondrial sub-populations. To address this issue, liver mitochondria from control and CR mice were fractionated using differential centrifugation at 1,000 g, 3,000 g and 10,000 g into distinct populations labeled as M1, M3 and M10, respectively. Mitochondrial protein levels, respiration and H(2)O(2) production were measured in each fraction. CR resulted in a decrease in total protein (mg) in each fraction, although this difference disappeared when adjusted for liver weight (mg protein/g liver weight). No differences in respiration (State 3 or 4) were observed between control and CR mice in any of the mitochondrial fractions. CR decreased H(2)O(2) production in all fractions when mitochondria respired on succinate (Succ), succ+antimycin A (Succ+AA) or pyruvate/malate+rotenone (P/M+ROT). Thus, CR decreased reactive oxygen species (ROS) production under conditions which stimulate mitochondrial complex I ROS production under both forward (P/M+ROT) and backward (Succ & Succ+AA) electron flow. The results indicate that CR decreases H(2)O(2) production in all liver mitochondrial fractions due to a decrease in capacity for ROS production by complex I of the electron transport chain.