Hypoxia initiates numerous intracellular signaling pathways important in regulating cell proliferation, differentiation, and death. In this study, we investigated the pathway that hypoxia uses to activate Akt and inactivate glycogen synthase kinase-3 (GSK-3), two proteins the functions of which are important in cell survival and energy metabolism. Severe hypoxia (0.01% oxygen) initiated a signaling cascade by inducing the tyrosine phosphorylation of the platelet-derived growth factor (PDGF) receptor within 1 h of treatment and increasing receptor association with the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). Hypoxia-induced signaling also resulted in PI 3-K-dependent phosphorylation of Akt on Ser-473, a modification of Akt that is important for its activation. This activation of Akt by hypoxia was substantially diminished in cells that possessed mutations in their PDGF receptor-PI 3-K interaction domain. In addition, Akt activation by hypoxia was resistant to treatment with the growth factor receptor poison suramin but was sensitive to treatment with the PI 3-K inhibitor wortmannin. Activation of Akt by hypoxia resulted in the phosphorylation of GSK-3alpha and GSK-3beta at Ser-9 and Ser-21, two well-documented Akt phosphorylation sites, respectively, that are inactivating modifications of each GSK-3 isoform. In support of the phosphorylation data, GSK-3 activity was significantly reduced under hypoxia. In conclusion, we propose that hypoxia activates a growth factor receptor/PI 3-K/Akt cascade that leads to GSK-3 inactivation, a pathway that can impact cell survival, proliferation, and metabolism.