Placental hypoxia likely plays an important role in both normal placental development and pathology. Yet, the molecular mechanisms of hypoxia signaling in this organ are virtually unexplored. Therefore, we investigated the expression of the hypoxia inducible transcription factors (HIF) in normal human placentas spanning the first trimester to term. Several key observations emerged: 1) HIF-1α and -2α mRNA were present in placentas of all gestational ages but with greater variability during early pregnancy; 2) overall, HIF-1α mRNA was expressed at a constant level in all placentas, whereas HIF-2α mRNA increased significantly with gestational age; 3) both HIF-1α and -2α protein decreased significantly with gestational age; and 4) HIF-1α and -2α immunoreactivity were overlapping in cellular distribution being expressed by the syncytiotrophoblast, villous cytotrophoblast, and fetoplacental vasculature with both nuclear and cytoplasmic localization. Next, we studied the regulation of these transcription factors by oxygen using placental villous explants in culture from first-trimester and term placentas. The major findings were 1) HIF-1α and -2α protein, but not mRNA, was induced by hypoxia in the placental villous explants; 2) HIF-1α DNA-binding activity was also stimulated by hypoxia; and 3) glucose transporter-1 mRNA (a known target of HIF) was also increased by hypoxia in placental villous explants. We suggest that physiological hypoxia contributes to the increased expression of HIF-1α and -2α protein in early placentas and that regulation of these transcription factors by hypoxia in the human placenta occurs at the level of protein and not mRNA.