Urokinase-type plasminogen activator (uPA) on prostate cancer cell surfaces mediates pericellular proteolysis and destruction of extracellular matrix barriers to tumor invasion and metastasis. Increased expression of tumor-associated uPA leads to enhanced tumor dissemination and poor cancer outcomes in men with prostate cancer. Expression of uPA is regulated in part by the oxidant-sensitive transcription factor, NF-kappa B (NF-kappaB), which is activated by intracellular reactive oxygen intermediates (ROI). This study examined the effect of iron on the production of ROI, activation of NF-kappaB and expression of uPA in the human prostate cancer cell line, PC-3. Treatment of PC-3 cells with iron in the form of ferric nitrilotriacetate (FeNTA) in the absence of added transferrin resulted in a dose-dependent increase in cellular ferritin content in both the presence and absence of neutralizing antibody to the transferrin receptor. Cellular uptake of iron resulted in stimulation of intracellular ROI production, and increases in uPA mRNA, antigen, and activity. Concurrent treatment with the iron chelator, desferrioxamine (DFO) abrogated these effects, and treatment with DFO alone inhibited constitutive uPA production. Finally, we observed nuclear translocation, and therefore activation of NF-kappaB in response to iron exposure. We conclude that iron enters PC-3 cells via a non-transferrin dependent pathway and increases uPA expression. Our data indicate that one mechanism by which iron may stimulate uPA production is through the generation of intracellular ROI and activation of NF-kappaB-mediated signaling pathways.