Soluble nickel compounds are carcinogenic to humans although the mechanism by which they cause cancer remains unclear. One major consequence of exposure to nickel is the stabilization of hypoxia inducible factor-1alpha (HIF-1alpha), a protein known to be overexpressed in a variety of cancers. In this study, we report a persistent stabilization of HIF-1alpha by nickel chloride up to 72 h after the removal of nickel from the culture media. In addition, we show that the HIF-prolyl hydroxylases (PHD's) are inhibited when cells are exposed to nickel and that they remain repressed for up to 72 h after nickel is removed. We then show that nickel can inhibit purified HIF-PHD's 2 in vitro, through direct interference with the enzyme. Through theoretical calculations, we also demonstrate that nickel may be able to replace the iron in the active site of this enzyme, providing a plausible mechanism for the persistent inhibition of HIF-PHD's by nickel. The data presented suggest that nickel can interfere with HIF-PHD directly and does not inhibit the enzyme by simply depleting cellular factors, such as iron or ascorbic acid. Understanding the mechanisms by which nickel can inhibit HIF-PHD's and stabilize HIF-1alpha may be important in the treatment of cancer and ischemic diseases.