In this study, the localization of plasminogen activator inhibitor-1 (PAI-1) and its association with glomerular subepithelial deposits of vitronectin (VN) in membranous nephropathy (MN) was evaluated. Renal biopsy tissue from 30 patients with MN along with specimens of normal kidney removed from six patients with renal tumors were examined for glomerular deposits of urokinase-type plasminogen activator, tissue-type plasminogen activator (t-PA), PAI-1, VN, and fibrinogen by using immunofluorescence and immunoelectron microscopic techniques. Deposits were characterized by pretreating the frozen sections with arginine, glycine, and t-PA to dissociate the VN-PAI-1 complexes, thus providing evidence of PAI-1 binding to VN. Glomeruli isolated from normal frozen kidney and from frozen kidney tissue with MN were subjected to immunoblot analysis. VN was found in the mesangial area and t-PA was observed along the capillary wall of normal glomeruli. Coarse granular deposits of VN, PAI-1, and t-PA were observed along the glomerular basement membrane in MN. Immunoelectron microscopy showed the presence of VN, PAI-1, and t-PA in the subepithelial immune deposits. Pretreating the samples with arginine decreased or abolished immunofluorescence staining for PAI-1 and t-PA, but not for VN. Pretreatment with t-PA decreased the immunofluorescence staining for PAI-1, but not for VN. Pretreatment with glycine decreased or abolished the staining for all three proteins. Immunoblot analysis revealed the presence of VN, PAI-1, t-PA, and t-PA-PAI-1 complex in glomeruli with MN as well as the presence of VN, but not of PAI-1, t-PA, or complex in the normal glomeruli. The findings from this study strongly suggest that PAI-1 was deposited in the glomeruli in MN as a result of binding to VN. Thus, VN-associated PAI-1 may injure the integrity of the fibrinolytic system in glomeruli affected by MN.