We previously reported that vascular endothelial growth factor (VEGF) increases vascular permeability through the synthesis of endothelial platelet‐activating factor (PAF), while others reported the contribution of nitric oxide (NO). Herein, we addressed the contribution of VEGF receptors and the role played by PAF and NO in VEGF‐induced plasma protein extravasation. Using a modified Miles assay, intradermal injection in mice ears of VEGF‐A₁₆₅, VEGF‐A₁₂₁, and VEGF‐C (1 µM) which activate VEGFR‐2 (Flk‐1) receptor increased vascular permeability, whereas a treatment with VEGFR‐1 (Flt‐1) analogs; PlGF and VEGF‐B (1 µM) had no such effect. Pretreatment of mice with PAF receptor antagonist (LAU8080) or endothelial nitric oxide synthase (eNOS) inhibitor (L‐NAME) abrogated protein extravasation mediated by VEGF‐A₁₆₅. As opposed to PAF (0.01‐1 µM), treatment with acetylcholine (ACh; up to 100 µM; inducer of NO synthesis) or sodium nitroprusside (SNP; up to 1 µM; NO donor) did not induce protein leakage. Simultaneous pretreatment of mice with eNOS and protein kinase A (PKA) inhibitors restored VEGF‐A₁₆₅ vascular hyperpermeability suggesting that endogenous NO synthesis leads to PKA inhibition, which support maintenance of vascular integrity. Our data demonstrate that VEGF analogs increase vascular permeability through VEGFR‐2 activation, and that both endogenous PAF and NO synthesis contribute to VEGF‐A₁₆₅‐mediated vascular permeability. However, PAF but not NO directly increases vascular permeability per se, thereby, suggesting that PAF is a direct inflammatory mediator, whereas NO serves as a cofactor in VEGF‐A₁₆₅ proinflammatory activities. J. Cell. Biochem. 100: 727–737, 2007. © 2006 Wiley‐Liss, Inc.