The estrogen receptor (ER) plays an important role in breast cancer development and progression. Hypoxia has been shown to modulate the level of ERalpha expression, which is intimately associated with the biology of breast carcinomas. However, the effect of hypoxia on ERalpha-mediated transactivation is largely unknown. In this report, we have examined ligand-independent transcriptional activation of ERalpha by hypoxia. The hypoxia-induced ERalpha-mediated transcriptional response was inhibited by the ER antagonist ICI 182,780 as determined by transient expression of ERalpha and ER-responsive reporter plasmids in the HEK 293 cells. Hypoxic activation of ERalpha was dependent on the increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha), as examined in HEK 293 cells under conditions of normoxia. These results indicate that hypoxia activates ERalpha in a ligand-independent manner, possibly through the interaction between HIF-1alpha and ERalpha.