Extracellular RNA and DNA are not inert molecules but contain biological activities. They may be released during cellular insults that initiate repair processes. In the present study the impact of t-RNA, whole cell RNA and DNA on vasculogenesis of mouse embryonic stem (ES) cells was investigated. It was observed that t-RNA and whole cell RNA but not DNA dose-dependent stimulated vasculogenesis of ES cells as assessed by CD31 immunohistochemistry. This was abolished in the presence of RNase. t-RNA treatment increased hypoxia-induced factor-1a (HIF-1a), vascular endothelial growth factor (VEGF) and neuropilin-1 (NRP-1) expression, and elevated reactive oxygen species (ROS) generation. ROS generation was inhibited in the presence of free radical scavengers as well as the NADPH oxidase inhibitors apocynin and VAS2870. The stimulation of vasculogenesis by t-RNA as well as increase in HIF-1a was abolished in the presence of the free radical scavengers N-(2-mercapto-propionyl)-glycine and trolox, indicating that vasculogenesis induced by t-RNA is regulated through ROS which are generated by NADPH oxidase.