During the growth of embryonic stem cells within the three- dimensional tissue of embryoid bodies, cells are subjected to mechanical strain which may represent an important stimulus for cell differentiation. In the present study embryoid bodies were cultivated on flexible membranes, static as well as cyclic mechanical strain was applied and endothelial cell as well as cardiac differentiation was investigated. Mechanical strain enhanced cardiomyogenesis as evaluated by the occurrence of spontaneously beating foci of cardiac cells, the size of cardiac areas, and mRNA expression of cardiac-specific genes. Furthermore, mechanical strain increased the area of PECAM-1- positive endothelial cells as well as hypoxia-inducible factor-1a?(HIF-1a) and vascular endothelial growth factor (VEGF). Mechanical strain increased the generation of reactive oxygen species (ROS) and the expression of NADPH oxidase subunits. Preincubation with free radical scavengers inhibited cardiovascular differentiation. Cardiomyogenesis was inhibited by the p38 inhibitor SB203580, the ERK1,2 inhibitor UO126, and the JNK inhibitor SP600125.Vasculogenesis/angiogenesis was blunted following inhibition of ERK1,2 and JNK, whereas p38 inhibition was ineffective. Our data outline a role of ROS as mechanotransducing molecules in mechanical strain-stimulated cardiovascular differentiation of embryonic stem cells.