The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its alpha subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1alpha by prolyl hydroxylases (PHDs) recruits the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the alpha subunit. Hypoxic stabilization of HIF-1alpha has been reported to be antagonized by nitric oxide (NO). By using a HIF-1alpha-pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1alpha by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1alpha stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1alpha protein stabilization. In vitro HIF-1alpha-pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1alpha destabilization by NO donors under hypoxia via modulation of PHD activity.