The isolated rat hepatocytes inoculated onto the surface of positively charged culture dishes are anchored initially and then begin to migrate and aggregate gradually to form multicellular spheroids detached from the dish. We studied the roles of fibrinolytic factors in the spheroid formation. The fibrinolytic factors, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), were increased in the course of spheroid formation. Then, we introduced fibrinolytic inhibitors into the spheroid cultures to determine functions of fibrinolytic factors. Plasmin inhibitor inhibited markedly the spheroid formation. Interestingly, the anti-plasmin antibody showed different effect depending on the timing of its administration. In summary, we demonstrated for the first time that induction of PAs and ensuing plasmin generation on the cell surface play important roles in hepatocyte spheroid formation, and that plasmin is involved in the different processes such as cell migration and cell detachment in the formation of hepatocyte spheroid.