Previous studies have shown that tissue fatty acid unsaturation correlates inversely with maximum longevity. However, it is unclear if this is related to the effects of fatty acid unsaturation only on lipids, or also on proteins and DNA, specially on mitochondrial DNA (mtDNA) oxidative damage. In this investigation the degree of fatty acid unsaturation of liver and brain was successfully manipulated in Wistar rats by chronic feeding with specially designed semipurified diets rich in saturated or unsaturated fats. The brain, an organ of special relevance for aging, was most profoundly affected by the increase in fatty acid unsaturation, and showed significant increases in malondialdehyde (MDA)-lysine, aminoadipic semialdehyde (a protein carbonyl), N(epsilon)-(carboxymethyl)lysine, and N(epsilon)-(carboxyethyl)lysine in proteins, as well as in 8-oxo,7,8-dihydro-2'-deoxyguanosine (8-oxodG) in mtDNA without changes in nuclear DNA (nDNA). In the liver 8-oxodG was also increased in mtDNA and not in nDNA. These DNA results are consistent with the presence of a high density of mitochondrial inner membranes (rich in lipids and in reactive oxygen species generation capacity) near mtDNA but not near nDNA. Among the protein markers analyzed, MDA-lysine was most consistent and responsive to fatty acid unsaturation, since it increased in both organs and showed the highest increase. These results, together with previous data from our laboratories, show that increasing the degree of fatty unsaturation of postmitotic tissues in vivo can raise not only lipid but also protein and mtDNA oxidative damage. This is mechanistically relevant in relation to the constitutively low tissue fatty acid unsaturation of long-lived animals.