Angiopoietin1 (Ang1) is a novel angiogenic factor with important actions on endothelial cell (EC) differentiation and vascular maturation. Ang1 has been shown to prevent EC apoptosis through activation of PI3-kinase/Akt, a pathway that is also known to activate endothelium nitric oxide synthase (eNOS). Therefore, we hypothesized that the angiogenic effects of Ang1 would also be dependent on the PI3-kinase/Akt pathway, possibly mediated by increased eNOS activity and NO release. Treatment of human umbilical vein endothelial cells with recombinant Ang1* (300 ng/ml) for 15 minutes resulted in PI3-kinase-dependent Akt phosphorylation, comparable to that observed with vascular endothelial growth factor (VEGF) (50 ng/ml), and increased NO production in a PI3-kinase/Akt-dependent manner. Capillary-like tube formation induced by Ang1* in fibrin matrix at 24 hours (differentiation index, DI: 13.74 ± 0.76 versus control 1.71 ± 0.31) was abolished in the presence of the selective PI3-kinase inhibitor, LY294002 (50 μmol/L) (DI: 0.31 ± 0.31, P < 0.01) or the NOS inhibitor, L-NAME (3 mmol/L) (DI: 4.10 ± 0.59, P < 0.01). In subcutaneous Matrigel implants in vivo, addition of recombinant Ang1* or wild-type Ang1 from conditioned media of COS-1 cells transfected with a pFLAG Ang1 expression vector, induced significant neovascularization to a degree similar to VEGF. Finally, angiogenesis in vivo in response to both Ang1 and VEGF was significantly reduced in eNOS-deficient compared with wild-type mice. In summary, our results demonstrate for the first time that endothelial-derived NO is required for Ang1-induced angiogenesis, and that the PI3-kinase signaling mediates the activation of eNOS and NO release in response to Ang1.