The interaction of 125I-labelled tissue-type plasminogen activator (125I-t-PA) with freshly isolated rat parenchymal and endothelial liver cells was studied. Binding experiments at 4°C with parenchymal cells and endothelial liver cells indicated the presence of 68000 and 44000 high-affinity t-PA-binding sites, with an apparent K(d) of 3.5 and 4 nM respectively. Association of 125I-t-PA with parenchymal cells was Ca2+-dependent and was not influenced by asialofetuin, a known ligand for the galactose receptor. Association of 125I-t-PA with liver endothelial cells was Ca2+-dependent and mannose-specific, since ovalbumin (a mannose-terminated glycoprotein) inhibited the cell association of t-PA. Association of 125I-t-PA with liver endothelial cells was inhibited by anti-(human mannose receptor) antiserum. Anti-(galactose receptor) IgG had no effect on 125I-t-PA association with either cell type. Degradation of 125I-t-PA at 37°C by both cell types was inhibited by chloroquine or NH4Cl, indicating that t-PA is degraded lysosomally. In vitro experiments with three monoclonal antibodies (MAbs) demonstrated that anti-t-PA MAb 1-3-1 specifically decreased association of 125I-t-PA with the endothelial cells, and anti-t-PA Mab 7-8-4 inhibited association with the parenchymal cells. Results of competition experiments in rats in vivo with these antibodies were in agreement with findings in vitro. Both antibodies decreased the liver uptake of 125I-t-PA, while a combination of the two antibodies was even more effective in reducing the liver association of 125I-t-PA and increasing its plasma half-life. We conclude from these data that clearance of t-PA by the liver is regulated by at least two pathways, one on parenchymal cells (not galactose/mannose-mediated) and another on liver endothelial cells (mediated by a mannose receptor). Results with the MAbs imply that two distinct sites on the t-PA molecule are involved in binding to parenchymal cells and liver endothelial cells. Chemicals/CAS: ammonium chloride, 12125-02-9; calcium, 7440-70-2; chloroquine, 132-73-0, 3545-67-3, 50-63-5, 54-05-7; immunoglobulin G, 97794-27-9; mannose, 31103-86-3, 3458-28-4; ovalbumin, 77466-29-6; tissue plasminogen activator, 105913-11-9; Antibodies, Monoclonal; galactose receptor; Galactose, 26566-61-0; Iodine Radioisotopes; mannose receptor; Mannose, 31103-86-3; Receptors, Cell Surface; Receptors, Immunologic; Tissue Plasminogen Activator, EC 3.4.21.68