Low levels of oxygen characterize certain biological settings such as the first trimester human placenta and rapidly growing tumors. Hypoxia in tumors and expression of high levels of tumor-associated plasminogen activator inhibitor-1 (PAI-1) indicate a poor prognosis for some cancer patients. Furthermore, PAI-1 may promote tumor invasion and metastasis by modulating cell adhesion and detachment from the extracellular matrix. In this study, we used immortalized human trophoblasts (HTR-8/SVneo cells) derived from first trimester placenta to study the effect of exposure to low levels of oxygen on PAI-1 expression. Cell viability following 24-h exposure to 1% oxygen was similar to that of cells cultured under 20% oxygen. Exposure to hypoxia resulted in time-dependent increases in PAI-1 mRNA and protein levels, as determined by Northern blot analysis and enzyme-linked immunosorbent assay. Culture with cobalt chloride or Tiron also resulted in increased PAI-1 mRNA levels, while carbon monoxide inhibited the hypoxia-mediated increase, thereby indicating that a heme protein is involved in the stimulation of PAI-1 expression by hypoxia. Incubation with transforming growth factor-β₁(TGF-β₁) also resulted in increased levels of PAI-1 mRNA. However, addition of a neutralizing anti-TGF-β antibody to hypoxic cultures did not abrogate the increase in PAI-1 mRNA levels, suggesting that hypoxia stimulates PAI-1 expression via a pathway that does not require TGF-β production. These results indicate that, through their effect on PAI-1 expression, oxygen levels may play an important role in modulating cellular migration and invasion.