Cardiomyocyte differentiation from embryonic stem (ES) cells has been achieved by using extrinsic factors such as growth factors, chemicals or physical factors. For an increasing group of pro-cardiomyogenic stimuli it has been demonstrated that cardiomyogenesis is regulated by endogenous ROS generation through NAD(P)H oxidases. This has been shown for mechanical stimulation, treatment with electromagnetic field pulses and incubation with cytokines and growth factors. NOX-1, NOX-2 and NOX-4 isoforms have been demonstrated to be expressed during ES cell differentiation and may be involved in cardiovascular differentiation events. In the present study it is evidenced that permanent lentiviral transfection with shRNA targets to silence the expression of specific NOX enzymes in ES cells resulted in inhibition of cardiomyogenesis. It is demonstrated that NOX-1, NOX-2 and NOX-4 are involved in signalling pathways stimulating cardiomyogenesis. Ascorbic acid, a strongly pro-cardiogenic agent, upregulated NOX-1 and NOX-4 expression and stimulated ROS, as well as nitric oxide (NO) generation. In early stages of differentiation (day1 - day4) ROS generation upon treatment with ascorbic acid prevailed in embryoid bodies, whereas during later stages of differentiation a decrease in ROS generation and an increase in NO generation was observed. Incubation of differentiating embryoid bodies with low concentrations of either H₂O₂ or the peroxynitrite donor 3-morpholino-sidnonimine (SIN-1) stimulated cardiomyogenesis to a comparable extent, suggesting an interaction of ROS and NO during ES cell-derived cardiomyogenesis. Consequently, inhibition of NO generation by the e-NOS inhibitor N(G)-amino-l-arginine (L-NAA) was without significant effect on cardiomyogenesis, whereas cardiomyogenesis was inhibited in the presence of the peroxynitrite scavenger N-(2-mercapto-propionyl)-glycine (NMPG). We conclude that NAD(P)H oxidases generating ROS supported by NO are the crucial regulators of cardiomyogenesis of ES cells.