Ascorbic acid (AA) has been previously shown to stimulate cardiomyogenesis of mouse embryonic stem cells. However, the underlying mechanisms are so far unknown. AA can be considered an efficient antioxidant, but it can not be ignored that under some circumstances, i.e. the presence of metal ions and adequate pH conditions, AA displays pro-oxidant and even mutagenic effects. In the present study an upregulation of the NADPH oxidases Nox-1 and Nox-4 upon incubation with AA was observed. The NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and apocynin abolished the effect of AA. The stimulation of cardiomyocyte differentiation by AA was significantly inhibited in the presence of the free radical scavengers ebselen and N2-mercaptopropionylglycin (MPG) as well as by DPI and apocynin. Furthermore, the ascorbic acid- induced upregulation of the cardiac-specific genes MLC2v, alpha- MHC, and ANF, as well as the cardiac-specific transcription factor Nkx-2.5 was significantly inhibited in the presence of apocynin. Expression levels of MLC2v as well as MLC2a were significantly decreased in samples treated with MPG. In summary, our data suggest that AA stimulates cardiomyogenesis of embryonic stem cells by involving a ROS-mediated signalling pathway which utilizes activation of NADPH oxidase.