To determine the prevalence and the characterization of antibodies to endothelial cells in patients with SSc, serum samples from 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 20 healthy control subjects were examined by ELISA using cultured human umbilical vein endothelial cells (HUVEC), indirect immunofluorescence analysis (IIF), and immunoblotting using cytoplasmic extract of HUVEC. IgG and/or IgM isotype anti-endothelial cell antibodies (AECA) were demonstrated by ELISA in 43 of 80 patients with SSc (54%), in 15 of 20 patients with SLE (75%), and in none of 20 healthy control subjects. Immunofluorescence analysis on HUVEC substrate showed homogeneous cytoplasmic staining. Immunoblotting demonstrated that these patients had antibodies directed to one or several antigens of approximately 60, 90, 110 and 140 kD, and the most common responses were to the 90-kD antigen. By the immunofluorescence method using HUVEC, affinity-purified anti-90-kD antibodies showed identical cytoplasmic staining to that produced by sera positive for AECA. Furthermore, AECA were closely correlated with pulmonary fibrosis in patients with SSc. These findings suggest that patients with SSc have abnormal antibodies to endothelial cell antigens, and support the hypothesis that endothelial dysfunction is involved in the development of this disease.