The amount of linoleic acid required to prevent undesirable effects of C18 trans fatty acids was investigated. In a first experiment, six groups of rats were fed diets with a high content of trans fatty acids (20% of energy [en%]), and increasing amounts of linoleic acid (0.4 to 7.1 en%). In a second experiment, four groups of rats were fed diets designed to compare trans fatty acids with saturated and cis-monounsaturated fatty acids of the same chain length at the 2 en% linoleic acid level. After 9-14 weeks, the oxygen uptake, lipid composition and ATP synthesis of heart and liver mitochondria were determined. The phospholipid composition of the mitochondria did not change, but the fatty acid compositions of the two main mitochondrial phospholipids were influenced by the dietary fats. Trans fatty acids were incorporated in all phospholipids investigated. The linoleic acid level in the phospholipids, irrespective of the dietary content of linoleic acid, increased on incorporation of trans fatty acids. The arachidonic acid level had decreased in most phospholipids in animals fed diets containing 2 en% linoleic acid. At higher linoleic acid intakes, the effect of trans fatty acids on the phospholipid arachidonic acid level diminished. However, in heart mitochondrial phosphatidylethanolamine, trans fatty acids significantly increased the arachidonic acid level. Despite these changes in composition, neither the amount of dietary linoleic acid nor the addition of trans fatty acids influenced the mitochondrial function. For rats, a level of 2 en% of linoleic acid is sufficient to prevent undesirable effects of high amounts of dietary C18 trans fatty acids on the mitochondrial function.