Human leucocyte antigen (HLA)-specific antibodies, present at the time of transplant, cause renal transplant rejection but cases of rejection of HLA-identical renal transplants indicate that antibodies to non-HLA antigens may also be detrimental. There is increasing evidence that antibodies to antigens present on endothelial cells and monocytes, and on endothelial cells alone, are associated with transplant rejection. We investigated 105 patients with failed renal transplants for the presence of endothelial cell reactive antibodies and compared them with 94 successful transplant patients to determine the role of non-HLA antibodies in transplant failure. Patient sera were tested by enzyme-linked immunoabsorbent assay (ELISA) using as a target fixed cells either from the endothelial/epithelial cell line EAHy.926 or primary cultures of human umbilical vein endothelial cells. Antibody binding was detected using an alkaline phosphatase-conjugated anti-human immunoglobulin G (IgG) antibody. Fourteen of the 105 failed transplant patients had endothelial cell-reactive antibodies as compared with only three of the 94 patients with successful transplants (Fisher's exact test, p = 0.02). Antibody-positive sera were absorbed with the epithelial cell line A549 to remove antibodies directed against the epithelial component of EAHy.926 and with a pool of lymphoblastoid cell line cells to remove HLA-specific antibodies. Absorption did not reduce antibody activity showing the antibodies to be directed against endothelial cell determinants. Antibody-positive sera were also tested by flow cytometry against the monocyte cell line THP-1 and 13 of the 14 patients were negative. In conclusion, we have demonstrated the presence of IgG antibodies directed against endothelial cell determinants in renal transplant recipients in association with renal transplant failure.