Endothelial cells (EC) express both hypoxia inducible factor-1alpha (HIF-1alpha) and -2alpha (HIF-2alpha), yet their roles in the EC hypoxic response are unclear. Hypoxia upregulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EC through a 5' hypoxic regulatory element (HRE). We compared the upregulation of GAPDH in human lung microvascular EC to that in hep3B cells, another cell type known to express both HIF-1alpha and HIF-2alpha. GAPDH mRNA increased to a lesser extent in hypoxic hep3B cells than in EC, yet upregulation occurred through the same HRE that was active in EC. HIF-1alpha protein induction in response to hypoxia was similar in both cell types. In contrast, HIF-2alpha protein levels were upregulated to a greater extent and for a longer period of time by hypoxia in EC than in hep3B cells. Correspondingly, electrophoretic mobility supershift assays showed that, in EC, there was preferential binding of HIF-2alpha to the GAPDH HRE while, in hep3B cells, there was binding of both HIF-1alpha and HIF-2alpha. The preferential binding of HIF-2alpha to the GAPDH HRE in EC may account for their higher level of induction of GAPDH. These findings suggest that cell-specific patterns of HIF-1alpha and HIF-2alpha expression lead to cell-specific gene upregulation during hypoxia.