In order to study the role of the asialoglycoprotein receptor (ASGPr) in the rapid plasma clearance of urokinase-type plasminogen activator (u-PA), a microtiter plate binding assay was developed using ASGPr purified from rat liver extracts. Urinary two-chain u-PA bound to immobilized ASGPr in a saturable manner with an EC50 of 0.2 microM. Binding was inhibited by rabbit antibodies against the ASGPr. In line with the known carbohydrate specificity of the ASGPr, GalNAc proved to be the most effective inhibitor from a series of monosaccharides, followed by Gal and Fuc, whereas GlcNAc was ineffective. The N-linked oligosaccharides of urinary u-PA do not terminate with the common Gal-GlcNAc element, but with a GalNAc-GlcNAc element which is partially sulfated. Sulfated forms of u-PA were separated from non-sulfated forms by using the lectin Wisteria floribunda agglutinin. Only the non-sulfated forms of u-PA (30% of the total) appeared to bind to the ASGPr. From different u-PA preparations used for thrombolytic therapy only urinary u-PA and u-PA produced by kidney cell cultures strongly bound to the ASGPr, whereas (recombinant) u-PA expressed in mouse myeloma cells, Chinese hamster ovary cells or E. coli scarcely bound to the receptor. It is concluded that u-PA bearing non-sulfated GalNAc-GlcNAc elements is specifically recognized by the ASGPr present on liver cells.