Cell injury leads to exposure of intracellular material and is associated with increased permeability of vessels in the vicinity of the damage. Here, we demonstrate that natural extracellular RNA as well as artificial RNA (poly-I:C), or single-stranded RNA but not DNA, significantly increased the permeability across brain microvascular endothelial cells in vitro and in vivo. RNA-induced hyperpermeability of tight monolayers of endothelial cells correlated with disintegration of tight junctions and was mediated through vascular endothelial growth factor (VEGF), reminiscent of heparin's activities. Antisense oligonucleotides against VEGF-receptor 2 (VEGF-R2) prevented the permeability-inducing activity of extracellular RNA and heparin completely. Hence, these polyanionic substances can lead to mobilization/stabilization of VEGF with the subsequent activation of VEGF-R2. In accordance with these functional data, strong binding of VEGF as well as other growth factors to RNA was demonstrable. In in vivo rat models of FeCl3-induced sinus sagittal is superior thrombosis and stroke/brain edema, pretreatment of animals with RNase (but not DNase) resulted in a significant reduction of vessel occlusion, infarct volume, and prevention of brain edema formation. Together, these results identify extracellular RNA as a novel natural permeability factor, upstream of VEGF, whereas counteracting RNase treatment may serve as new vessel-protective modality.