Macrophage Migration Inhibitory Factor (MIF) is an important proinflammatory cytokine involved in regulation of macrophage function. In addition, MIF may also play a role in murine and human reproduction. While both frist trimester trophoblast and decidua express MIF, the regulation and functional significance of this cytokine during human placental development remains unclear. We assessed MIF expression throughout normal human placental development, as well as in in vitro (chorionic villous explants) and in vivo (high altitude placentae) models of human placental hypoxia. Dimethyloxalylglycine (DMOG), which stabilizes Hypoxia Inducible Factor-1 (HIF-1) under normoxic conditions, was also used to mimic the effects of hypoxia on MIF expression. Quantitative RT-PCR and Western blot analysis showed high MIF protein and mRNA expression at 7-10 weeks, and lower levels at 11-12 weeks until term. Exposure of villous explants to 3% O₂ resulted in increased MIF expression and secretion relative to standard conditions (20% O₂). DMOG-treatment under 20% O₂ increased MIF expression. In situ hybridization and immunohistochemistry showed elevated MIF expression in low oxygen-induced extravillous trophoblast cells. Finally, a significant increase in MIF transcript was observed in placental tissues from high altitude pregnancies. Hence three experimental models of placental hypoxia (early gestation, DMOG-treatment, and high altitude) converge in stimulating increased MIF, supporting the conclusion that placental-derived MIF is an oxygen-responsive cytokine highly expressed in physiological in vivo and in in vitro low oxygen conditions.