The occurrence of autoimmune phenomena in 28 patients with thromboangiitis obliterans (Buerger's disease) was determined. The following were sought: antineutrophil cytoplasmic antibodies against proteinase 3 (cANCA) and myeloperoxidase (pANCA), antinuclear antibodies, anti-Ro antibodies, anticardiolipin antibodies, and antiendothelial cell antibodies (AECA). For the last, an enzyme-linked immunosorbent assay was developed which verified the presence of the AECA phenomenon with immunofluorescence and confocal microscopy. Seven patients with active disease had AECA titers of 1,857± 450 arbitrary units (AU) compared with 126 ± 15 AU in 30 normal control subjects (P < 0.001) and 461 ± 41 AU in 21 patients in remission (P < 0.01). Antibodies from the sera of patients with active disease reacted not only with surface epitopes but also with sites within the cytoplasm of human endothelial cells. AECA may be useful in following disease activity and may play a role in the pathogenesis of thromboangiitis obliterans.