In order to obtain a routine simple screening test for the detection of anti-endothelial antibodies (AEA) we developed a highly reproducible and sensitive enzyme-linked immunosorbent assay (ELISA) on fixed endothelial hybridoma cells. Detection of AEA with this type of monolayer appeared to be superior to ELISAs with monolayers of human umbilical vein endothelial cells, unfixed endothelial hybridoma cells or assays with membranes of endothelial cells. Glutaraldehyde treated endothelial hybridoma cells are most appropriate for use in ELISA procedures to detect AEA because the endothelial hybridoma cells are easy to culture, form a constant antigenic source and when plated and fixed to microtitre plates they can be stored without losing their ability to bind AEA.