We examined how ionizing radiation (IR) delivered under either severe hypoxia (< 0.1% O₂) or normoxia affects the expression of hypoxia inducible factor 1α (HIF-1α) and the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins 1, 2 and 4 in U87 human glioblastoma cells. IR was delivered as single doses of 0, 2, 5, 10 and 20 Gy after 6-hr hypoxic incubation and in normoxic controls. Irradiation at any dose did not affect the cellular protein levels of any of the angiopoietins, whereas hypoxia led to increasing levels of both angiopoietin-4 and angiopoietin-2. Levels of angiopoietin-1 protein were unaltered throughout the observation period. A dose-dependent increase in levels of secreted VEGF in the medium occurred after IR at doses from 5-20 Gy. In hypoxic cells, 20 Gy IR induced an additional significant increase in VEGF relative to nonirradiated hypoxic control cells with elevated baseline VEGF levels induced by hypoxia. HIF-1α and glucose transporter-1 (Glut-1) were not correspondingly upregulated by IR. Blocking HIF-1α by antisense treatment induced a reduced baseline VEGF at normoxia, while the relative upregulation of VEGF by IR was unaffected. These data provide evidence that VEGF is upregulated by IR by mechanisms independent of HIF-1 transactivation.