Previously, we have reported that eNOS promoter activity is decreased in pulmonary arterial endothelial cells (PAECs) in response to hydrogen peroxide (H₂O₂). Thus, the objective of this study was to identify the cis-element(s) and transcription factor(s) responsible for oxidant-mediated downregulation of the eNOS gene. Initial promoter experiments in PAECs treated with H₂O₂ revealed a significant decrease in activity of a promoter fragment containing 840 bp of upstream sequence of the human eNOS gene fused to a luciferase reporter. However, a promoter construct containing only 640 bp of upstream sequence had a significantly attenuated response to H₂O₂ challenge. As the 840 bp promoter construct had a putative binding site for the transcription factor AP-1 that was lacking in the 640bp construct, we evaluated the effect of H₂O₂ on promoter activity after mutation of the AP-1 binding sequence (TGAGTCA at -661 to TGAGTtg in the 840 bp construct). Similar to the results seen with the 640 bp, the AP-1 mutant promoter had a significantly attenuated response to H₂O₂. Electrophoretic mobility shift assay revealed decreased binding of AP-1 during H₂O₂ treatment. Supershift analysis indicated that the AP-1 complex consisted of a c-Jun and FosB heterodimer. Further, in vitro EMSA analysis indicated the c-Jun binding was significantly decreased after H₂O₂ exposure. Using chromatin immunoprecipitation analysis we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to H₂O₂. These data suggest a role of decreased AP-1 binding likely through c-Jun, in the H₂O₂ mediated decreased in eNOS promoter activity.