Corneas used for transplantation are typically obtained from donors up to 48 hr post mortem. By this time, standard HLA typing usually is impossible because of the lack of viable lymphocytes in spleen and peripheral blood. To increase the number of HLA-typed corneas, we developed a method in which the retinal pigment epithelial (RPE) cells of the donor eye are isolated, cultured in the presence of 1000 IU/ml interferon-gamma (IFN-gamma), and, after 4 d, are typed for HLA Class I and Class II antigens in the standard NIH cytotoxicity assay. Sixty five donors were typed simultaneously using peripheral blood lymphocytes and RPE. One hundred and sixteen out of 120 HLA-A antigens, 125/127 HLA-B, 106/108 HLA-C, and 92/100 HLA-DR antigens were identical using the same technique. Donor age, sex, cause of death, time of enucleation post mortem, and time of RPE preparation post mortem, as well as duration of culture period prior to stimulation with IFN-gamma did not correlate with the results of HLA typing. These data show that RPE cells can substitute for lymphocytes in post mortem HLA typing. Consequently, every donor with corneas suitable for transplantation can be prepared for matched transplantation.