Autoimmunity to glomerular basement membrane (GBM) could induce Goodpasture disease, and natural autoantibodies against GBM in the sera of normal individuals were not reported. The aim of the study was to identify and characterize natural autoantibodies against GBM in normal human sera. Natural anti-GBM autoantibodies were purified from the sera of five healthy persons by affinity chromatography, using purified bovine alpha(IV)non-collagenous (NC1) as solid-phase ligands. Antigen specificity, immunoglobulin G (IgG) subclasses, and antibody avidity of the natural autoantibodies were investigated by enzyme-linked immunosorbant assay (ELISA), Western-blot analysis, indirect immunofluorescence, and antigen-inhibition ELISA, and compared with those of 32 patients with anti-GBM disease. Natural anti-GBM autoantibodies could be purified from IgG fractions of all the five persons, with an average amount of 0.5% of total IgG fractions. Antigen specificity of the natural autoantibodies was identified by blotting to human alpha(IV)NC1, reactivity to recombinant alpha3(IV)NC1, and linear staining along the GBM of normal kidney sections. Titers of the natural autoantibodies were much lower than those of patients (1:60.6 vs 1:993.6, P<0.001). IgG subclasses distribution of the natural autoantibodies was restricted to IgG2 (100%) and IgG4 (100%), while for patients it was mainly IgG1 (93.8%) and IgG4 (90.6%). Avidity of the natural autoantibodies was lower than that of patients, the amount of alpha(IV)NC1 used for 50% inhibition was 1.65 and 0.46 microg, respectively (P<0.05). In conclusion, natural anti-GBM autoantibodies exist in normal human sera. Antibody levels, IgG subclasses, and avidity of the natural autoantibodies were different from those of patients. Fine specificity of the natural autoantibodies needed to be elucidated.