Endothelial cells demonstrate high urokinase expression and upregulation of urokinase receptors in response to vascular injury. Urokinase receptor binding facilitates endothelial cell migration into an arterial wound; however, the signaling cascade induced by the urokinase receptor in this cell type is incompletely understood. Because the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway seems to be important for vessel function, we investigated the hypothesis that urokinase receptor binding activates Jak/Stat signaling in human vascular endothelial cells. Incubation of endothelial cells with urokinase-type plasminogen activator (uPA,1 nmol/L) induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with a molecular weight between 80 to 90 and 130 to 140 kDa. The same pattern of tyrosine phosphorylation was found after treatment with 1 nmol/L ATF, the urokinase amino-terminal fragment, which is devoid of proteolytic activity but still binds to the urokinase receptor. Using coimmunoprecipitation techniques, we demonstrated that the activated urokinase receptor is associated with 2 cytoplasmic tyrosine kinases of the Jak family, viz, Jak1 and Tyk2. uPA and ATF induced a time-dependent activation of both kinases, as shown by immunoprecipitation and Western blot analysis. Using electrophoretic mobility shift and supershift assays, we then demonstrated that Stat1 is rapidly activated in endothelial cells in response to uPA and ATF. Furthermore, Stat1 specifically binds to the regulatory elements interferon-gamma activation site/interferon-stimulated response element. The uPA-induced, time-dependent translocation of Stat1 to cell nuclei was confirmed by confocal microscopy study and immunoblotting of nuclear extracts with an anti-Stat1 antibody. This study provides evidence for a novel signaling pathway for uPA in human vascular endothelial cells. Direct activation of the Jak/Stat system via the uPA-receptor complex may be an important mechanism for endothelial cell migration and/or proliferation during angiogenesis and after vascular injury.