The present study demonstrates that natural IgG with anti-endothelial cell activity is present in the serum of healthy individuals and in pooled normal human Ig. By using a novel method that allows for the simultaneous and quantitative assessment of reactivities of antibodies with a large number of antigens in tissues, we observed that natural anti-endothelial cell antibody (AECA) recognizes a restricted set of self antigens in endothelial cells that is conserved among healthy individuals. The extent to which natural AECA activity is expressed in serum and the pattern of reactivity of AECA with endothelial cell antigens showed little variability between individuals. Analysis of AECA in the serum of patients with systemic lupus erythematosus (SLE) revealed a higher amount of activity and a wider spectrum of antigenic specificities than that recognized by natural antibodies in endothelial cell extracts. AECA activity of IgG in whole serum was lower than that of purified IgG in the case of healthy individuals and showed little variation among individuals. In contrast, no difference was found between AECA activity of purified IgG and that of IgG in patients' serum suggesting that SLE sera lack the factors that control expression of AECA activity in the serum of healthy individuals. Our results indicate that natural autoantibodies recognize a restricted and conserved set of self antigens. Our observations further suggest that defective regulation of the expressed autoreactive B cell repertoire is the basis for expansion of novel clonal specificities and enhanced autoantibody activity in serum of patients with autoimmune disease.