Cardiomyogenesis in differentiating mouse embryonic stem (ES) cells is promoted by cardiotrophin-1 (CT-1). Chemical (CoCl₂) and physiological (1% O2) hypoxia increased CT-1, HIF-1aprotein and mRNA expression as well as reactive oxygen species (ROS) levels in embryoid bodies. Treatment of cardiac cells from ES cells with CoCl₂ resulted in nuclear translocation of CT-1, increased gp130 phosphorylation and elevated protein expression of NADPH-oxidase subunits p22-phox, p47-phox, p67-phox, as well as Nox-1 and Nox-4 mRNA. Consequently, inhibition of NADPH-oxidase activity by diphenylen iodonium chloride (DPI) and apocynin abolished pro-oxidant- and chemical hypoxia-induced up-regulation of CT-1. CoCl₂ activated ERK1,2, JNK and p38 as well as PI3-kinase. The CoCl₂-dependent up-regulation of CT-1 was inhibited in the presence of the ERK1,2 antagonist UO126, the JNK antagonist SP600125, the p38 antagonist SKF86002, the PI3-kinase antagonist LY294002, the Jak-2 antagonist AG490 as well as in the presence of free radical scavengers. Moreover, developing embryoid bodies derived from HIF-1a-/--ES cells lack cardiomyogenesis, and pro-oxidants as well as chemical hypoxia failed to up-regulate CT-1 expression. Our data demonstrate that CT-1 expression in ES cells is regulated by ROS and HIF-1 and imply a crucial role of CT-1 in survival and proliferation of ES cell-derived cardiac cells.