Previous comparative studies have shown that long-lived animals have lower fatty acid double bond content in their mitochondrial membranes than short-lived ones. In order to ascertain whether this trait protects mitochondria by decreasing lipid and protein oxidation and oxygen radical generation, the double bond content of rat heart mitochondrial membranes was manipulated by chronic feeding with semi-purified AIN-93G diets rich in highly unsaturated (UNSAT) or saturated (SAT) oils. UNSAT rat heart mitochondria had significantly higher double bond content and lipid peroxidation than SAT mitochondria. They also showed increased levels of the markers of protein oxidative damage malondialdehyde-lysine, protein carbonyls, and N(e)-(carboxymethyl)lysine adducts. Basal rates of mitochondrial oxygen radical generation were not modified by the degree of fatty acid unsaturation, but the rates of H2O2 generation stimulated by antimycin A were higher in UNSAT than in SAT mitochondria. These results demonstrate that increasing the degree of fatty acid unsaturation of heart mitochondria increases oxidative damage to their lipids and proteins, and can also increase their rates of mitochondrial oxygen radical generation in situations in which the degree of reduction of Complex III is higher than normal. These observations strengthen the notion that the relatively low double bond content of the membranes of long-lived animals could have evolved to protect them from oxidative damage.