Here, we demonstrate by chromatin immunoprecipitation that the binding of hypoxia-inducible factors to gene regulatory regions is differentially influenced in cancer cells. Binding of HIF-2 alpha varies depending on hypoxic conditions, although HIF-1 alpha is constantly bound to these regions. We found by RNA interference experiments that HIF-2 alpha plays a minor role in VEGF gene upregulation under hypoxia or CoCl2 treatment, even when both HIFs are similarly bound to the promoter region. HIF-2 alpha activated or suppressed the ENO1 gene under various conditions, irrespective of promoter binding. We additionally found that HIF dependence on EPO gene induction could be altered depending on the conditions, irrespective of the binding pattern of HIFs. These results demonstrate that, unlike HIF-1 alpha, HIF-2 alpha differentially binds and regulates transcription under hypoxia. (C) 2008 Elsevier Inc. All rights reserved.