A growth factor stimulating DNA synthesis of adult rat hepatocytes in primary culture was found in the conditioned medium after culturing nonparenchymal liver cells (NPC). Adding heparin to the NPC cultured medium stimulated the growth-factor secretion from NPC. The growth factor was secreted mainly by Kupffer cells. The partially purified growth factor from the NPC appeared to be related to the HGF isolated from platelets according to three criteria: (a) binding to Heparin-Sepharose and eluting at about 0.65 M NaCl, (b) having a Mr of about 70 kDa, (c) having an immunoreactivity to antibody against rat platelet-derived HGF. Adding heparin to the NPC cultured medium also resulted in protection of the growth factor from heat- and acid-inactivation, but the direct interaction of heparin with the partially purified growth factor did not lead to such protection. Perfusion of normal adult rat livers with Hanks' solution containing 1 M NaCl in situ led to the release of large amounts of hepatocyte growth factor (10). These findings suggest that the hepatocyte growth factor derived from NPC binds to ECM between the layer of hepatocytes and endothelial cells forming the sinusoids in normal adult rat liver and that this may play a role in stabilization and maintaining the pool of HGF, which functions to constantly supply HGF in the setting of liver regeneration.