Age-related damage to the mitochondrial membrane, including decreased membrane fluidity, has been attributed to free radical reactions. Our previous studies point to lipid peroxidation as a primary cause in age-related changes in membrane fluidity. This report offers new evidence that lipid peroxidation-modulated decreases in membrane fluidity are mediated through two aldehydic lipid peroxidation products, 4-hydroxynonenal (HNE) and malondialdehyde (MDA). Hepatic mitochondria were isolated from both ad libitum fed (AL) and dietary restricted (DR) rats of different ages. Introduction of the aldehydes was found to decrease mitochondrial membrane fluidity, although the fluidity decrease induced by HNE was more pronounced than that induced by MDA. It seems likely that HNE modifies membrane fluidity by direct interaction with membrane phospholipids, as shown by the generation of a fluorescent complex between HNE and membrane phospholipids. Finally, HNE and MDA were isolated and quantitated in mitochondria. Their levels clearly differentiated between animals of different age and dietary groups. These data indicate that the reactive products of lipid peroxidation, especially HNE, may play an important role in mediating the decreased mitochondrial membrane fluidity observed in aging animals.