The phosphatidylinositol 3-kinase (PI3K) signaling pathway has inherent oncogenic potential. It is up-regulated in diverse human cancers by either a gain of function in PI3K itself or in its downstream target Akt or by a loss of function in the negative regulator PTEN. However, the complete consequences of this up-regulation are not known. Here we show that insulin and epidermal growth factor or an inactivating mutation in the tumor suppressor PTEN specifically increase the protein levels of hypoxia-inducible factor (HIF) 1α but not of HIF-1β in human cancer cell lines. This specific elevation of HIF-1α protein expression requires PI3K signaling. In the prostate carcinoma-derived cell lines PC-3 and DU145, insulin- and epidermal growth factor-induced expression of HIF-1α was inhibited by the PI3K-specific inhibitors LY294002 and wortmannin in a dose-dependent manner. HIF-1β expression was not affected by these inhibitors. Introduction of wild-type PTEN into the PTEN-negative PC-3 cell line specifically inhibited the expression of HIF-1α but not that of HIF-1β. In contrast to the HIF-1α protein, the level of HIF-1α mRNA was not significantly affected by PI3K signaling. Vascular endothelial growth factor reporter gene activity was induced by insulin in PC-3 cells and was inhibited by the PI3K inhibitor LY294002 and by the coexpression of a HIF-1 dominant negative construct. Vascular endothelial growth factor reporter gene activity was also inhibited by expression of a dominant negative PI3K construct and by the tumor suppressor PTEN.