The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) have extensive structural homology and have been identified as transcription factors that mediate hypoxia-inducible gene expression through hypoxia-responsive element (HRE). They play critical roles not only in normal development, but also in tumor progression. Endothelial cells (EC) express both HIF-1alpha and -2alpha. In this study, we examined the subcellular localization of HIF-1alpha and -2alpha in bovine arterial EC (BAEC) by immunoblotting and immunocytostaining analysis and found that even under normoxic conditions, as with its heterodimeric partner ARNT, HIF-2alpha was stable, and was localized in the nucleus of BAEC differently than HIF-1alpha. HIF-2alpha might be regulated by a different mechanism than HIF-1alpha and might mediate the expression of some EC-specific genes under normoxic conditions. We further found that cardiovascular helix-loop-helix factor (CHF) 2, which had been identified as an ARNT-interacting protein, was expressed in BAEC and suppressed HRE-dependent gene expression both under normoxia and hypoxia. CHF2 might be one of the key regulators of HIFalpha-mediated gene expression in normoxic EC. (C) 2004 Elsevier Inc. All rights reserved.