We have previously reported that oral administration of allogeneic rat spleen cells before kidney allotransplantation significantly prolongs graft survival. This prolongation was alloantigen specific and was associated with a decrease in graft-infiltrating cells (GIC) and an increase in transcription of IL-4 mRNA in the GIC. In this study increased splenic mixed lymphocyte responses from animals orally exposed to alloantigen before kidney transplantation suggested that the kidney allograft prolongation was not due to a masking of allorecognition, but to an immunomodulation of the immune response. We have assessed GIC T cell subsets on day 5 post-transplant and found decreased numbers of CD4(+) T cells in fed animals compared with controls, but there was no change in CD8(+) T cell numbers. The CD8(+) GIC from fed animals transcribed substantial levels of perforin, granzyme, and Fas ligand mRNA, indicating the presence of active CTL. Direct CTL assays showed that the GIC from fed recipients exhibited higher allo-CTL activity than GIC from control unfed recipients. In addition, the CD8(+) GIC exhibited high levels of IL-4 mRNA, suggesting Tc2-type regulatory cells. Prolonged graft survival in the face of active CTL and Tc2 cells suggests the presence of a CD8(+) regulatory cell population in the allograft. To confirm this, cell transfer experiments were performed. Prolongation of graft survival was transferred from rats orally exposed to alloantigen to naive animals by transfer of CD8(+) GIC. This is the first report that oral exposure to alloantigen prolongs kidney allograft survival by the generation of intragraft CD8(+) regulatory cells.