Critical to the success of human corneal transplants is prevention of corneal endothelial rejection, yet little is known about the endothelial infiltrate. To examine the endothelium, a method for removal and processing this layer as a flat sheet was used and the infiltrate was compared with stroma and epithelium. LEW or PVG strain rat corneas were transplanted to PVG strain recipients. Clinical changes after transplantation were monitored by slit lamp and animals sacrificed at a range of time points during rejection. Clinically defined rejection, accompanied by an epithelial rejection line and endothelial cell infiltration, occurred between days 10 and 15. There was some infiltration of leukocytes in the stroma of isografts at these time points, but significantly more in allografts (p < 0.003 for all subsets). There was no infiltration of isograft endothelium at any time and no infiltration of allograft endothelium on day 10. On day 15, there were similar numbers of all major subsets except B cells in the stroma, while on the endothelium macrophages, MHC class II+ cells and CD8+ cells predominated (p < 0.001 CD4+ vs CD8+ cells). T cells and NK cells predominated in the epithelial rejection line. While TNF-α and IFN-γ-producing cells were numerous in stroma and epithelium, no IFN-γ-producing cells were found on endothelium. Distinct differences in infiltrative profile within layers of the cornea suggest that the mechanisms of rejection may also differ. The restricted endothelial cell profile and lack of IFN-γ suggests that the anti-endothelial response may be modulated by the anterior chamber environment.