Anti-endothelial and other cell membrane-reactive antibodies in scleroderma were characterized by immunoblotting sera with membrane and cytosol preparations of human umbilical vein endothelial cells (HUVEC), dermal fibroblasts and a T cell lymphoma HUT78. Antibodies reactive with HUVEC membranes were found in 17 of 20 patients with scleroderma (33 bands) in contrast to only two of 20 controls (two bands; P < 0.01) and three of 11 patients with myocardial infarction (four bands). Eleven of the 20 patients possessed antibodies that were specific for HUVEC membrane and did not cross-react with other cell lines. Analysis of patient subgroups showed that HUVEC membrane antibodies were present in nine of 11 patients with systemic sclerosis and in all nine with the CREST syndrome, and were HUVEC-specific in five and six of these cases, respectively. Although considerable heterogeneity was seen, antibodies to an 18-19-kD membrane epitope were found in 11 of the 20 patients but in none of the controls (P < 0.01). This antibody which reacted particularly with HUVEC (n = 9) and HUT78 membranes (n = 9) was associated with CREST syndrome rather than systemic sclerosis (9/9 versus 1/11; P < 0.01), and after elution was shown to possess anticentromere activity. In addition, antibodies reactive with both fibroblast (n = 11; 18 bands) and HUT78 membranes (n = 18; 42 bands) were detected and were specific for either fibroblast or HUT78 membranes in nine and 14 patients, respectively. There was no significant difference in the incidence of these fibroblasts and HUT78 membrane antibodies in the two patient subgroups. These findings support the concept that membrane-reactive antibodies, including anticentromeric antibodies, may play a central role in the pathogenesis of scleroderma, through their ability to react with endothelial cells.