Impaired fibrinolytic activity within the lungs is a common manifestation of acute and chronic inflammatory lung diseases. Our previous work using transgenic mice showed that upregulation of fibrinolysis reduced pulmonary fibrosis following bleomycin-induced inflammatory lung injury. As a strategy to accelerate fibrinolysis, we generated recombinant adenoviruses containing human and mouse urokinase-type plasminogen activator (uPA) cDNAs. Both vectors induced the expression of functional uPA in human lung-derived epithelial A549 cells. A single intratracheal instillation of these uPA-containing adenoviruses into mouse lungs resulted in increased plasminogen activator activity in bronchoalveolar lavage fluid for at least 2 weeks. Plasma-derived fibrin-rich matrices overlaid on A549 cells infected with these uPA vectors were lysed efficiently in a dose-dependent fashion. Similarly, fibrin matrices formed within intact lungs that had been infected with these uPA-containing adenoviruses were also lysed more rapidly compared with noninfected and control virus-infected lungs. These results indicate that adenovirus-mediated transduction of uPA successfully upregulates fibrinolysis in vitro and in vivo. These uPA vectors can be readily used for testing the role of the fibrinolytic system in animal models of lung fibrosis, with particular attention to their therapeutic potential.