Hypoxia inducible factor-1 (HIF-1) is a heterodimeric transcription factor that regulates expression of several hypoxia-inducible genes, including erythropoietin (EPO), by binding to hypoxia response elements (HREs) in their promoters/enhancers. Previously, we have shown that 17-β estradiol (E2-β) attenuates hypoxic induction of EPO in rats. We hypothesized that this response is mediated by E2-β-induced attenuation of HIF-1α activity/expression. To test this hypothesis, we performed reporter gene assays in Hep3B cells to assess E2-β effects on hypoxia-induced activity of a reporter gene driven by the HRE from a cloned EPO-enhancer element. Immunocytochemistry and Western blots were additionally used to determine effects of E2-β on hypoxic increases in HIF-1α and EPO immunoreactivity. Finally, we examined potential influences of E2-β on HIF-1α mRNA levels by real-time PCR. Consistent with our hypothesis, E2-β (100 pM) inhibited hypoxic increases in HRE-mediated reporter gene activity. Furthermore, the estrogen-receptor antagonist ICI 182,780 (25 μM) eliminated these inhibitory effects of E2-β. E2-β similarly attenuated hypoxic induction of both EPO and HIF-1α protein in an estrogen-receptor dependent manner, but was without effect on HIF-1α mRNA expression. These findings suggest a role for E2-β to attenuate EPO expression by interfering with hypoxic increases in HIF-1α protein through an estrogen receptor-dependent mechanism.