Aims: We investigated the role of superoxide O2– during the initiation of vascular endothelial growth factor (VEGF)- and placental growth factor (PlGF)-mediated signal transduction in bone marrow-derived endothelial cells. Methods: BMhTERT cells were treated with VEGF or PlGF in the presence or absence of antioxidants. The signaling pathways downstream were analyzed by immunoprecipitation and Western blotting. Superoxide and reactive oxygen species (ROS) were measured using Superluminol or 2′,7′-dichlorofluorescein fluorescence measurements. Results: We show here that VEGF and PlGF generate extracellular and intracellular O2– that regulates their downstream signaling transduction pathways. Indeed, the extracellular O2– generated treatment of endothelial cells (using hypoxanthine/xanthine oxidase) was sufficient to initiate receptor phosphorylation of VEGF receptor 2. The PlGF treatment of endothelial cells increased the generation of intracellular ROS in an extracellular O2– dependent manner. Quenching of intracellular ROS by resveratrol inhibits PlGF- and VEGF-dependent induction of MAP kinase phosphorylation. Additionally, we found that the interaction of VEGF and PlGF with their specific receptors generates O2– in a cell-free system. Endothelial cells treated with VEGF stop proliferation in the presence of extracellular catalase, superoxide dismutase or peroxiredoxin IV. Conclusion: Our studies underscore the role of O2– as a critical regulator of VEGF and PlGF signal transduction initiation in endothelial cells.