The hypoxia-inducible transcription factor-1 (HIF-1) consists of an inducible a-subunit and a constitutively expressed b-subunit. Under normoxia, HIF-1a is hydroxylated by prolyl hydroxylases (PHD) within the N-terminal transactivation domain (N-TAD) and by asparagine hydroxylation within the C-terminal TAD by the hydroxylase factor inhibiting HIF (FIH), preventing p300/CBP binding. Since we demonstrated that HIF-1a accumulates in response to thrombin and H2O2 in smooth muscle cells, we investigated the role of these stimuli in the regulation of HIF-1a hydroxylation. Hypoxia, thrombin and H2O2 induced HIF-1a levels and activity whereas vitamin C (VitC)prevented it. Two hybrid assay in which a construct containing Gal4 response elements was cotransfected with N-TAD or C-TAD fused to Gal4-DNA binding domain showed that these stimuli increased luciferase activity and were unefficient when proline (in N-TAD) and asparagine (in C-TAD) were mutated. All stimuli reduced the interaction with pVHL but was restored by VitC pretreatment. p300/CBP binding to HIF-1a was enhanced by stimulation and prevented by VitC. Thus, thrombin and H2O2-induced oxidative stress lead to enhanced HIF-1 activity by inhibiting PHD and FIH activity in VSMC. Since thrombin and oxidative stress have been implicated in many vascular diseases targeting the PHD/FIH/HIFpathway may provide new therapeutics.