A critical event in tumor development is the formation of new blood vessels to provide oxygen, nutrients and growth factors to the rapidly growing cancer cells. This process of angiogenesis is complex, however, it is well established that vascular endothelial growth factor (VEGF)-mediated signaling is an important early event. Knockout mice studies have implicated the EP3 receptor in tumor development and angiogenesis; however, the signaling mechanism involved with this effect is unclear. We now show that stimulation of the EP3(I) isoform of the human EP3 receptor with prostaglandin E(2) increases the mRNA expression of both VEGF and its cognate receptor VEGF receptor-1 (VEGFR-1). These inductions by the EP3(I) receptor involve the sequential activation of phosphatidylinositol 3-kinase and the extracellular signal-regulated kinases. Up-regulation of VEGF and VEGFR-1 mRNA by the human EP3(I) receptor has not been previously reported and further strengthen the role of this receptor in tumor-associated angiogenesis.