Tissue plasminogen activator (tPA) has a prominent role in physiological fibrinolysis in vivo. Thrombosis has been associated with clinical scenarios (e.g., atherosclerotic disease) known to involve local decreases in tPA activity with concomitant formation of reactive nitrogen species such as peroxynitrite (OONO-), a molecule formed from nitric oxide and superoxide. We hypothesized that exposure of tPA to OONO- would result in a decrease in tPA activity. OONO- was generated with 3-morpholinosydnonimine (SIN-1), a molecule that produces both nitric oxide and superoxide. Recombinant tPA was incubated at 37°C for 60 min with 0 μM SIN-1; 100 μM SIN-1; 100 μMSIN-1 and 4000 U/mL recombinant human superoxide dismutase; or 4000 U/mL recombinant human superoxide dismutase (n = 8 separate reactions per condition). Changes in tPA activity were assessed by addition of tPA samples to tissue factor-exposed human plasma and measuring clot fibrinolysis with a thrombelastograph®. Exposure to SIN-1 resulted in a decrease in tPA-mediated fibrinolysis (<1% activity of tPA not exposed to SIN-1) that was significantly (P < 0.001) different from the other three conditions. There were no significant differences between the other conditions. We conclude that tPA is inhibited by OONO-, and that OONO - may have a role in clinical thrombotic scenarios.