Integrin a3ß1 is a major receptor for laminin. The expression levels of laminins-8 and -10 in the basement membrane surrounding blood vessels are known to change during tumor angiogenesis. Although some studies have suggested that certain ligands of a3ß1 can affect angiogenesis either positively or negatively, either a direct in vivo role for a3ß1 in this process or its mechanism of action in endothelial cells during angiogenesis is still unknown. Because the global genetic ablation of a3-integrin results in an early lethal phenotype, we have generated conditional-knockout mice where a3 is deleted specifically in endothelial cells (ec-a3-/-). Here we show that ec-a3-/- mice are viable, fertile, and display enhanced tumor growth, elevated tumor angiogenesis, augmented hypoxia-induced retinal angiogenesis, and increased vascular endothelial growth factor (VEGF)-mediated neovascularization ex vivo and in vivo. Furthermore, our data provide a novel method by which an integrin may regulate angiogenesis. We show that a3ß1 is a positive regulator of endothelial-VEGF and that, surprisingly, the VEGF produced by endothelial cells can actually repress VEGF-receptor 2 (Flk-1) expression. These data, therefore, identify directly that endothelial a3ß1 negatively regulates pathological angiogenesis and implicate an unexpected role for low levels of endothelial-VEGF as an activator of neovascularization.