Tumor hypoxia and high levels of expression of the urokinase-type plasminogen activator (uPA) receptor (uPAR) represent a poor clinical outcome for patients with various cancers. Here, we examined the effect of hypoxia on in vitro invasion of extracellular matrix and uPAR expression by human carcinoma cells. Compared with culture under 20% O2, culture for up to 24 hr under 1% or 4% O2 resulted in increased cell surface uPAR. However, the highest uPAR levels were observed in cells cultured under 1% O2. Culture of MDA-MB-231 breast carcinoma cells under hypoxia also resulted in increased uPAR mRNA levels. Furthermore, incubation with cobalt chloride or with an iron chelator also resulted in elevated uPAR expression, while presence of 30% carbon monoxide in the hypoxic atmosphere reduced the hypoxia-mediated uPAR mRNA upregulation. Increased uPAR expression was paralleled by higher cell-associated uPA levels and lower levels of secreted uPA as determined by gel zymography performed on cell extracts and culture-conditioned media. In addition, the in vitro invasiveness of MDA-MB-231 breast carcinoma cells was significantly higher when the invasion assay was performed under hypoxic conditions. This effect of hypoxia on invasion was abrogated by including in the assay a monoclonal, function-blocking anti-u PAR antibody or by the presence of 30% carbon monoxide in the hypoxic atmosphere. Our findings indicate that hypoxia stimulates carcinoma cell invasiveness by upregulating uPAR expression on the cell surface through a mechanism that requires a putative heme protein. Through a similar mechanism, hypoxia may stimulate tumor invasion and metastasis in vivo.