To determine the clinical significance of anti-endothelial cell antibodies in mixed connective tissue disease (MCTD), we measured the titers of antibodies to both untreated and cytokine-treated endothelial cells (EC) in the sera of the MCTD patients by means of an enzyme linked immunosorbent assay. The mean titer of antibodies to untreated EC (aEC) in the sera of the MCTD patients was significantly higher than that for the healthy subjects. The mean titer of antibody to EC treated with IFN gamma, IL1 (aIL1-EC) or TNF alpha (aTNF-EC) was significantly higher than that of aEC in the sera of the MCTD patients with proteinuria, and the mean titer of aTNF-EC was significantly higher than that of aEC in the sea of the MCTD patients with pulmonary fibrosis. Furthermore, the mean titers of aIL1-EC and aTNF-EC in the sera of the MCTD patients with pulmonary fibrosis were significantly higher than those of aIL1-EC and aTNF-EC in the sera of the MCTD patients without pulmonary fibrosis. These results suggest that antibodies to cytokine-treated EC may play a more important role in the manifestation of renal or pulmonary lesions in MCTD patients than aEC.