Tumor cells respond to hypoxic stress by upregulating a variety of genes involved in glucose uptake, glycolysis, and angiogenesis, all essential to maintaining nutrient availability and intracellular ATP levels. Adenosine monophosphate-dependent kinase (AMPK) is a key sensor for cellular homeostasis and is highly sensitive to changes in AMP:ATP ratios. The two catalytic AMPK alpha isoforms (AMPKα1, AMPKα2) were investigated with respect to their expression, cellular distribution, and contribution to VEGF expression under hypoxic stress in human U373 glioblastoma cells. Quantitative real-time PCR analysis showed AMPKα1 mRNA to be constitutively expressed in normoxia and hypoxia, whereas AMPKα2 mRNA levels were low in normoxia and significantly induced in hypoxia. Fluorescent immunohistochemistry showed that AMPKα2 protein redistributed to the nucleus under hypoxia, whereas AMPKα1 remained distributed throughout the cell. The AMPK chemical inhibitor, 5-iodotubericidin, effectively repressed the hypoxic induction of VEGF mRNA levels and hypoxia inducible factor-1 dependent transcription. AMPKα2 repression with RNA interference reduced hypoxia-induced VEGF mRNA and HIF-1 transcription, whereas AMPKα1 repression did not. Human glioblastoma cell lines U118 and U138 also showed hypoxia-induction of AMPKα2 as well as VEGF. Immunohistochemistry analysis of human astrocytoma/glioma samples revealed AMPKα2 present in high grade gliomas within hypoxic pseudopalisading microenvironments. These data suggest that prolonged hypoxia promotes the expression and functional activation of AMPKα2 and VEGF production in glioma cell lines and glioblastoma multiform tumors, thus contributing to tumor survival and angiogenesis in high grade human gliomas. © 2006 Wiley-Liss, Inc.