Hepatocyte growth factor (HGF), also known as scatter factor, is believed to play a primary role in liver regeneration. HGF is produced in an inactive single-chain form that can be cleaved in vitro to the active two-chain form by tissue-type and urokinase-type plasminogen (PLG) activators (tPA and uPA). We have now documented the de novo appearance of active uPA in livers from male Fischer F344 rats that underwent 70% partial hepatectomy (PHx) as early as 1 minute after surgery. Western blot analyses of protein extracts from liver remnants that were obtained immediately after surgery and periodically until 24 hours after PHx indicate that the quantity of uPA remains fairly constant in PHx samples. In contrast, the uPA receptor (uPAR) dramatically increases, beginning within 1 minute after PHx. This results in enhanced activity of uPA, as seen by direct zymography on cryostat sections. The uPA present in remnant liver homogenates from rats that underwent PHx is the primary agent that cleaves single-chain HGF to its two-chain form, because cleavage can be prevented when antibody against uPA is included in the liver homogenates. Furthermore, heterodimeric HGF, which is not present in normal liver, increases in the liver remnants from rats that underwent PHx, correlative to uPAR. The presence of active uPA is one of the earliest responses yet documented after PHx. These findings imply that both uPA and uPAR are involved in activating endogenous HGF in the regenerating livers of animals that underwent PHx.