The peroxisome proliferator-activated receptor α (PPARα) is a transcription factor belonging to the PPAR subfamily of nuclear receptors. Fatty acids and eicosanoids are natural PPARα ligands. Here, we show using transient transfection assays that oxidized (oxLDL) but not native low-density lipoproteins (LDL) dose-dependently activate PPARα in endothelial cells without affecting PPARα protein expression. Fractioning of oxLDL lipids followed by transactivation experiments demonstrated that the oxidized phospholipid component in oxLDL is responsible for PPARα activation. Using specific inhibitors, it is shown that oxLDL-mediated PPARα activation requires phospholipase A2 activity and that the oxidized fatty acids 9- and 13-HODE activate PPARα directly. Finally, we found that, similar to the synthetic PPARα ligand Wy-14643, oxLDL induced expression of the fatty acid transport protein-1 in human primary endothelial cells. Our findings define a novel group of PPARα activators and provide a molecular basis for certain effects of these biologically active phospholipids on gene transcription.