Urokinase-type plasminogen activator (uPA) and its specific receptor (uPAR) act in concert to stimulate cytoplasmic signaling machinery and transcription factors responsible for cell migration and proliferation. Recently we demonstrated that uPA activates the Janus kinase/signal transducers and activators of transcription (Stat1) signaling in human vascular smooth muscle and endothelial cells. However, the important question whether other transcription factors of the Stat family, in addition to Stat1, are involved in the uPAR-related signaling has not been addressed. In this study, we demonstrate that Stat4 and Stat2, but not Stat3, Stat5, or Stat6, are rapidly activated in response to uPA. We demonstrate further that Stat4 and Stat2 rapidly and transiently translocate to the cell nucleus where they bind specifically to the regulatory DNA elements. Analysis of Stat complexes formed in response to uPA revealed a Stat2-Stat1 heterodimer, which lacks p48, a DNA-binding protein known to combine with Stat1-Stat2. This new uPA-induced Stat2-Stat1 heterodimer binds to GAS (the interferon-gamma activation site) distinct from the interferon-stimulated response element to which the p48 protein containing complexes generally bind. We conclude that uPA activates a specific and unusual subset of latent cytoplasmic transcription factors in human vascular smooth muscle cells that suggests a critical role of uPA in these cells.