Endothelial cells in various tissues of the body are often exposed to hypoxic conditions. To examine the effects of sustained hypoxia on energy metabolism in endothelial cells, we have maintained bovine aortic and human umbilical vein endothelial cells in an atmosphere containing low oxygen concentrations (14 mmHg) for up to 96 h. We report here that endothelial cells maintained under these conditions upregulate their glucose transport activity, consume more glucose, and produce greater amounts of lactic acid than normoxic cells. Upregulation of glucose transport activity by hypoxic endothelial cells required several hours to occur, was associated with increased expression of mRNA and protein for the erythroid/brain form of the facilitative glucose transporter, and was not due to depletion of glucose from the medium. Prolonged treatment of endothelial cells with inhibitors or uncouplers of oxidative phosphorylation (antimycin, azide, dinitrophenol) under normoxic conditions also upregulated glucose transporter expression. These results suggest that reduced rates of oxidative metabolism may represent an important signal for cells to adapt metabolically to hypoxia. Furthermore, in our examination of endothelial cell energy metabolism, we discovered that endothelial cells contain phosphocreatine and express both the brain and muscle isozymes of creatine kinase.