Peroxisome proliferator-activated receptors (PPARa, -b and -[gamma]) are nuclear receptors involved in transcriptional regulation of lipid metabolism. The aim of this study was to investigate the role of PPARa in cardiomyogenesis during the differentiation of mouse embryonic stem (ES) cell- derived embryoid bodies (EBs). When EBs were treated with PPARa agonists (WY14643, GW7647 and Ciprofibrate) a significant increase in cardiomyogenesis and expression of the cardiac genes MLC2a, ANP, MHC-ß, MLC2v, and cardiac a-actin was observed. In contrast, the PPARa antagonist MK886 decreased the number of beating foci. The effect of the PPARa agonists was abolished when EBs were pre-incubated with the free radical scavengers Vitamin E (trolox) and N-(2-mercapto-propionyl)-glycine, indicating the involvement of reactive oxygen species (ROS). Furthermore, we observed an increase in ROS when EBs were treated with PPARa agonists, and consequently a decrease in intracellular ROS when EBs were treated with MK886. The effect of PPARa agonists on intracellular ROS was attenuated by the NADPH-oxidase inhibitors DPI and apocynin, indicating the involvement of NADPH oxidase. In summary our data indicate, that PPARa stimulation induces cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase.