Fetal bovine aortic endothelial GM 7373 cells were transfected with a viral expression vector harboring the human urokinase-type plasminogen activator (uPA) gene. The stable transfectant clone uPA-R5 overexpressed and secreted human uPA as shown by Northern blot analysis, immunoprecipitation of metabolically labeled proteins, plasmin chromogenic assays, and SDS-PAGE zymography of cell extracts and conditioned media. The uPA-R5 cells were analyzed for their invasive capacity in vitro in the Matrigel chemoinvasion assay in the presence of serine- or metalloprotease inhibitors. uPA overexpression enhanced the invasive capacity of GM 7373 cells through a mechanism which differs from that mediated by metalloproteases. Endothelial cell uPA transfectants may represent an useful experimental model to investigate the role of uPA in angiogenesis and angioproliferative diseases.