Male rats of the Wistar strain were given oleuropein for 3 weeks at a dose of 25 or 50 mg/kg of body weight. Heart samples were analyzed for the lipid composition by the Iatroscan technique and for the fatty acid profile of neutral and polar lipids by the capillary gas chromatography. In addition, the oleuropein, α- and β-tocopherol content in the total heart lipids were also determined. Oleuropein treatment increased the level of cholesteryl esters, but it decreased the level of triacylglycerols, in the heart of animals. Sphingomyelin and lysophosphatidylcholine levels were only higher in the 50 mg oleuropein group. There was also a significant reduction in the linoleic acid content in the heart polar lipids of oleuropein-treated animals. As consequence of treatment, oleuropein was incorporated into the heart of animals in a concentration-dependent manner, whereas the α- and β-tocopherol content decreased. Oleuropein may therefore act as a substitute of the natural antioxidants in heart, with minor changes in the unsaturated lipids.