After birth mammalian cardiomyocytes slow down their proliferation rate, and heart growth is overtaken by cell hypertrophy. Herein, it is shown that low levels of reactive oxygen species (ROS) stimulated cardiomyogenesis of embryonic stem (ES) cells and induced cell proliferation of ES cell-derived cardiomyocytes as investigated by nuclear translocation of cyclin D1, downregulation of p27^KIP1 , phosphorylation of retinoblastoma protein, increase of Ki67 expression, and incorporation of BrdU. The observed effects were blunted in the presence of free radical scavengers. Furthermore, treatment with ROS increased expression of the cardiac-specific transcription factors GATA-4, Nkx-2.5, MEF2C, and DTEF-1 as well as of the cardiomyogenesis-associated growth factor BMP-10. Upon treatment with ROS increased expression of NADPH-Oxidase subunits Nox-1, Nox-4, p22-phox, p47-phox and p67-phox was observed, indicating feed-forward regulation of ROS generation. Consequently, the induction of cardiomyogenesis was inhibited in the presence of the NADPH-oxidase inhibitors DPI and apocynin. In summary, our data suggest that cardiomyogenesis of ES cells involves ROS-mediated signalling cascades.