Reactive oxygen species (ROS) are used as second messengers in response to many growth and differentiation stimuli incriminated in hypertensive pulmonary vascular remodeling. We found that a surprising target of ROS generated during signaling in pulmonary vascular endothelial and smooth-muscle cells was DNA in the nucleus and the mitochondria. The distribution of oxidative modifications between the genomes differed according to the initiating stimulus. Whereas the receptor-mediated agonists thrombin, platelet-derived growth factor, and angiotensin II increased equilibrium lesion density in both mitochondrial DNA and the nuclear vascular endothelial growth factor (VEGF) gene, hypoxia, which may elicit mitochondrial ROS production, modified only the VEGF gene. Interestingly, all three stimuli increased lesion density in the VEGF promoter. When modifications were mapped at single nucleotide resolution, the most frequently targeted nucleotide was a guanine located at the 3′ end of the hypoxia-inducible factor-1 DNA recognition sequence. Introduction of an abasic site at the oxidatively modified guanine enhanced incorporation of hypoxia-inducible factor-1 in the transcription complex forming on an oligonucleotide corresponding to the hypoxic response element of the VEGF gene and engendered more robust reporter gene expression. ROS generated in the context of physiologic signaling may threaten genomic integrity and thereby play a role in the microsatellite instability and somatic mutation detected in endothelial cells involved in plexiform lesion formation.1 Additionally, physiologically related DNA base modifications may comprise a previously unappreciated mechanism regulating genes incriminated in hypertensive pulmonary vascular remodeling.