Vascular endothelial growth factor (VEGF) plays an essential role in normal uterine physiology and function as well as endometrial cancer and other uterine disorders. Recently we showed that estrogen regulation of VEGF expression in the rat uterus involves rapid recruitment of both estrogen receptor (ER)-alpha and hypoxia-inducible factor (HIF)-1alpha to the VEGF promoter. Estrogen is known to stimulate both the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways, which have been linked to the activation of both of these transcription factors. Therefore, the involvement of these pathways in estrogen-induced VEGF expression was investigated. Inhibitors of the MAPK (U0126) or PI3K pathways (wortmannin or LY294002) were administered ip to immature female rats 1 h before 17beta-estradiol (E(2)) treatment. E(2) activation of both pathways occurred and was completely inhibited by the appropriate antagonist. Only PI3K inhibitors, however, blocked E(2) stimulation of VEGF mRNA expression and E(2)-induced uterine edema. In vivo chromatin immunoprecipitation analysis showed that this was associated with a failure of both HIF-1alpha and ERalpha to bind to the VEGF promoter. To determine whether inhibiting the PI3K pathway affected ERalpha induction of other estrogen target genes, the expression of creatine kinase B and progesterone receptor A/B was also examined. The expression of each was also inhibited by wortmannin, as was ERalpha binding to the creatine kinase B promoter. In conclusion, although estrogen activates both the MAPK and PI3K pathways in the rat uterus, activation of HIF-1alpha and ERalpha, and therefore regulation of VEGF gene expression is dependent only on the PI3K/Akt pathway. Furthermore, activation of the PI3K pathway appears to be a common requirement for the expression of estrogen-induced genes. These findings not only shed light on estrogen action in normal target tissues but also have important implications for cancer biology because excessive PI3K, HIF-1alpha, and VEGF activity are common in estrogen-dependent tumors.