Angiogenesis is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Targeted disruption of several EphB class receptor tyrosine kinases results in vascular remodeling defects during embryogenesis. The role of EphA class receptors in vascular remodeling, however, is not well-characterized. We recently demonstrated that global inhibition of EphA receptors disrupts endothelial migration induced by ephrin, VEGF or tumor-derived signals, though the specific target remained undefined. Here, we report that EphA2 regulates endothelial cell assembly and migration through phosphoinositide (PI) 3-kinase-mediated activation of Rac1 GTPase in two model systems: primary bovine and murine pulmonary microvascular endothelial cells. EphA2-deficient endothelial cells fail to undergo vascular assembly and migration in response to ephrin-A1 in vitro. Ephrin-A1 stimulation induces PI3-kinase-dependent activation of Rac1 in wild-type endothelial cells, whereas EphA2-deficient cells fail to activate Rac1 upon stimulation. Expression of dominant negative PI3-kinase or Rac1 inhibits ephrin-A1-induced endothelial cell migration. Consistent with in vitro data, EphA2-deficient mice show a diminished angiogenic response to ephrin-A1 in vivo. Moreover, EphA2-deficient endothelial cells fail to assemble in vivo when transplanted into recipient mice. These data suggest that EphA2 is an essential regulator of post-natal angiogenesis.