The proteolytic cascade involving plasminogen activators and plasmin appears to have an important function in tissue regeneration. We have investigated the expression and cellular localization of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) as well as plasminogen activation in rat liver regeneration by recruitment of progenitor (oval) cells. Using a model in which surgical partial hepatectomy is combined with feeding of 2- acetylaminofluorene (2-AAF) to induce liver regeneration by proliferation and differentiation of oval cells, expression of uPA, uPAR, and PAI-1 was detected by immunohistochemistry mainly in the duct-like formations of expanding oval cells. Plasminogen activation, as assessed by direct zymography on frozen liver sections, was located over the expanding oval cell populations but not over mature hepatocytes. Plasminogen activation was not detected in control liver. Expression of uPA, uPAR, and PAI-1, as assessed by immunohistochemical and Northern blot analyses, was also observed, when cells located in and in close proximity to the bile epithelial structures were activated to enter DNA-synthesis in response to 2-AAF, and after in vivo infusion of various growth factors. Given the physiologic function of plasminogen activation in fibrinolysis, and plasminogen activators in activation of latent growth factors, the selective expression of the plasminogen activator/plasmin proteolytic cascade in oval cells expanding during liver regeneration in response to the combination of 2-AAF and partial hepatectomy, may confer a proliferative advantage to these cell populations in an extracellular matrix containing both fibrin and latent growth factors.