Hypoxia inducible factor-1alpha (HIF-1alpha) is a proangiogenic transcription factor stabilized and activated under hypoxia. It regulates the expression of numerous target genes, including vascular endothelial growth factor (VEGF) and other cytoprotective proteins. In this study, we hypothesized that bone marrow stem cells (BMSCs) secrete growth factors which protect cardiomyocytes via HIF-1alpha pathway. BMSCs were obtained from transgenic mice overexpressing green fluorescent protein (GFP). The study was carried out in vitro using co-culture of BMSCs with cardiomyocytes. LDH release, MTT uptake, DNA fragmentation and annexin-V positive cells were used as cell injury markers. The level of HIF-1alpha protein as well as its activated form and VEGF were measured by ELISA. The expression of HIF-1alpha and VEGF in BMSCs was analyzed by quantitative PCR and cellular localization was determined by immunohistochemistry. LDH release was increased and MTT uptake was decreased after exposure of cardiomyocytes to hypoxia for 30 h, which were prevented by co-culturing cardiomyocytes with BMSCs. Cardiomyocyte apoptosis induced by hypoxia and H(2)O(2) was also reduced by co-culture with BMSCs. VEGF release from BMSCs was significantly increased in parallel with high level of HIF-1alpha in BMSCs following anoxia or hypoxia in a time-dependent manner. Although no significant up-regulation could be seen in HIF-1alpha mRNA, HIF-1alpha protein and its activated form were markedly increased and translocated to the nucleus or peri-nuclear area. The increase and translocation of HIF-1alpha in BMSCs were completely blocked by 2-methoxyestradiol (2-ME2; 5 mumol), a HIF-1alpha inhibitor. Moreover, the protection of cardiomyocytes by BMSC and VEGF secretion was abolished by neutralizing HIF-1alpha antibody in a concentration dependent manner (200-3200 ng/ml). Bone marrow stem cells protect cardiomyocytes by up-regulation of VEGF via activating HIF-1alpha.