The vascular endothelium plays a critical role in the response of cancer to ionizing radiation. Activation of the phosphoinositide-3-kinase/Akt pathway is one initial signaling event in irradiated endothelial cells. Specifically, a low dose of ionizing radiation (3 Gy) induces phosphorylation of Akt at Ser473 in the vascular endothelium within minutes of irradiation. However, signaling events that are downstream of Akt have not been well defined. Here, we show that phosphorylation of the Akt downstream target glycogen synthase kinase-3beta (GSK-3beta) at Ser9 also occurred within minutes of exposure to ionizing radiation. In addition, ionizing radiation caused the dissociation of GSK-3beta from the cell membrane, consistent with the inactivation of GSK-3beta enzyme activity. Overexpression of the dominant negative mutant Akt attenuated GSK-3beta phosphorylation at Ser9 and enhanced radiation-induced apoptosis. X-irradiated endothelial cells formed capillaries in both in vitro and in vivo models, whereas overexpression of the dominant negative mutant Akt inhibited capillary tubule formation. Studies using GSK-3beta antagonists showed that GSK-3beta activity was required for apoptosis in endothelial cells treated simultaneously with Akt antagonists and radiation. In mouse vascular models, radiation-induced microvascular destruction in response to Akt antagonists also required GSK-3beta function. These data indicate that on exposure of vascular endothelium to ionizing radiation, activation of Akt signaling contributes to GSK-3beta inhibition, which in turn promotes endothelial cell survival and capillary formation. Thus, pharmacologic regulation of Akt/GSK-3beta signaling may present a new approach to the radiation response in the tumor microvasculature.