In the absence of accessory components, plasminogen activator inhibitor 1 (PAI-1) rapidly forms equimolar, inactive complexes both with tissue-type (t-PA) and with urokinase-type (u-PA) plamsinogen activator. In the presence of either the glycoprotein vitronectin or the glycosaminoglycan heparin, PAI-1 is endowed with additional, efficient thrombin-inhibitory properties (Ehrlich et al., 1990, 1991a). Here, we have investigated the interaction between PAI-1, thrombin, and glycosaminoglycans in more detail. Inhibition of thrombin by PAI-1 was quantitatively analyzed in the presence of a wide range of concentrations of heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chrondoitin 6-sulfate, keratan sulfate, and hyaluronic acid by measuring residual amidolytic activity. In addition, a qualitative analysis was performed by determining the formation of SDS-stable, equimolar complexes between thrombin and PAI-1 in the presence of various glycosaminoglycans. Heparin, at concentrations between 0.1 and 1 µg/mL, significantly promoted thrombin inhibition by PAI-1 as well as SDS-stable complex formation. Suboptimal inhibition was observed with dermatan sulfate, chondroitin 4-sulfate, and heparan sulfate at concentrations that are at least 1 order of magnitude higher than that required for optimal inhibition in the presence of heparin. Virtually no inhibition of thrombin and SDS-stable complex formation was detected with any of the other glycosaminoglycans at concentrations between 0.1 and 1 µg/mL. Competition experiments between solid-phase heparin and soluble glycosaminoglycans for binding to metabolically labeled 35S-PAI-1 revealed a half-maximal inhibition value (IC50) of 4 µg/mL for heparin and approximately 200 µg/mL for dermatan sulfate and chondroitin 4-sulfate, whereas values larger than 200 µg/mL were found for the other glycosaminoglycans, supporting the notion that the ability to sustain thrombin inhibition is due to binding of the inhibitor. Partial depolymerizaton of heparin, followed by chromatographic size fractionation of different heparin species, allowed the determination of the number of monosaccharide units required for efficient promotion of both thrombin inhibition and SDS-stable complex formation between thrombin and PAI-1. Species below about 14 monosaccharide units did not promote thrombin inhibition and the formation of SDS-stable complexes. Optimal inhibition of thrombin by PAI-1 and promotion of SDS-stable complex formation were achieved with fractionated high molecular weight heparin. Finally, a dye displacement assay was used to establish difference spectra of heparin/proflavine vs proflavine in the presence of either thrombin or PAI-1. The displacement of the dye by thrombin, but not by PAI-1, indicated that the protease and the inhibitor occupy different binding sites on heparin. Our results are consistent with a template mechanism for the assembly of thrombin and PAI-1 on high molecular weight heparin.