Long-chain fatty acids (FA) coordinately induce the expression of a panel of genes involved in cellular FA metabolism in cardiac muscle cells, thereby promoting their own metabolism. These effects are likely to be mediated by peroxisome proliferator-activated receptors (PPARs). Whereas the significance of PPARα in FA-mediated expression has been demonstrated, the role of the PPARβ/δ and PPARγ isoforms in cardiac lipid metabolism is unknown. To explore the involvement of each of the PPAR isoforms, neonatal rat cardiomyocytes were exposed to FA or to ligands specific for either PPARα (Wy-14,643), PPARβ/δ (L-165041, GW501516), or PPARγ (ciglitazone and rosiglitazone). Their effect on FA oxidation rate, expression of metabolic genes, and muscle-type carnitine palmitoyltransferase-1 (MCPT-1) promoter activity was determined. Consistent with the PPAR isoform expression pattern, the FA oxidation rate increased in cardiomyocytes exposed to PPARα and PPARβ/δ ligands, but not to PPARγ ligands. Likewise, the FA-mediated expression of FA-handling proteins was mimicked by PPARα and PPARβ/δ, but not by PPARγ ligands. As expected, in embryonic rat heart-derived H9c2 cells, which only express PPARβ/δ, the FA-induced expression of genes was mimicked by the PPARβ/δ ligand only, indicating that FA also act as ligands for the PPARβ/δ isoform. In cardiomyocytes, MCPT-1 promoter activity was unresponsive to PPARγ ligands. However, addition of PPARα and PPARβ/δ ligands dose-dependently induced promoter activity. Collectively, the present findings demonstrate that, next to PPARα, PPARβ/δ, but not PPARγ, plays a prominent role in the regulation of cardiac lipid metabolism, thereby warranting further research into the role of PPARβ/δ in cardiac disease.