Cardiotypic development in embryonic stem cell-derived embryoid bodies may be regulated by reactive oxygen species (ROS). ROS were generated by a NADPH oxidase-like enzyme which was transiently expressed during the time course of embryoid body development. Incubation with either H(2)O(2) or menadione enhanced cardiomyogenesis, whereas the radical scavengers trolox, pyrrolidinedithiocarbamate and N-acetylcysteine exerted inhibitory effects. The phosphatidylinositol 3-kinase (PI-3-kinase) inhibitors LY294002 and wortmannin abolished cardiac commitment and downregulated ROS in embryoid bodies. Coadministration of LY294002 with prooxidants resumed cardiomyocyte differentiation, indicating a role for PI-3-kinase in the regulation of the intracellular redox state.