Cyto-ELISA has been widely used to investigate anti-endothelial cell antibodies (AECAs); however, because various types of endothelial cells have been used, the results among studies differ. The aim of our study was to analyze and compare the results when determining AECAs in patients with connective tissue disease (CTD). We did so using a cyto-ELISA with different cells as antigenic substrates: two different endothelial cells, one microvascular (HMEC-1) and one from human bone marrow (HBMEC), and one epithelial cell line from breast adenocarcinoma as negative controls (MDA-MB-231). In this trial, we performed a retrospective study in 60 patients with CTD [46 with systemic lupus erythematosus, 8 with Sjögren's syndrome, and 6 with systemic sclerosis] and 32 healthy volunteers. Using cyto-ELISA, the antibody against a cell was considered positive when the optical density (OD) obtained was higher than the mean OD obtained in the control group + 2 standard deviations (upper normal range). Patients were classified into three groups according to the OD obtained with the different cell lines: group 1: patients without any antibody; group 2: patients with specific AECAs; and group 3: patients with nonspecific AECAs. According to this classification, we found that 43.3% of patients with CTD have specific AECAs, and 28.3% have nonspecific antibodies. Our study delineates the heterogeneity of AECAs in patients with CTD. The use of HBMEC in cyto-ELISA may increase the sensitivity of the test, and the use of nonendothelial cells as negative controls may improve its specificity.