Mitochondria from liver whole homogenates of fischer 344 rats 3,6,12 and 24 mo of age were separated by reorienting rate-zonal centrifugation on sucrose gradients. Employing this method, mitochondria can be harvested nearly quantitatively and subjected to a number of biochemical analyses. Separation and recovery of the mitochondria from young and old rats was monitored by assaying each fraction from the gradient for cytochrome oxidase and lipoamide dehydrogenase, two mitochondrial specific enzymes. In addition, several regions of the gradient were studied by transmission electron microscopy. In this study, mitochondrial DNA and protein were quantitated from the livers of the animals at each age group. It was demonstrated that between the ages of 12 and 24 mo these macromolecules decreased significantly. These data would seem to indicate that the number of mitochondria is decreased in this tissue in aged animals.