The role of the hematopoietic lineage-restricted minor histocompatibility (H) antigen HA-1 in renal allograft tolerance was explored. We obtained peripheral blood samples from three recipients of histocompatibility leukocyte antigen (HLA)-matched, HA-1-mismatched renal transplants, one of which had discontinued immunosuppression >30 yr ago while sustaining normal kidney function. Peripheral blood mononuclear cells (PBMCs) were injected into the footpads of severe combined immunodeficiency mice to measure human delayed type hypersensitivity (DTH) responses. All three patients manifested regulated DTH responses to HA-1H peptide. By differential tetramer staining intensities, we observed two distinct minor H antigen HA-1-specific CD8 + T cell subsets. The one that stained dimly had the characteristics of a T regulatory (TR) cell and produced interleukin (IL) 10 and/or transforming growth factor (TGF) β. These HA-1-specific TR cells coexisted with bright tetramer-binding CD8+ T effector (T E) cells. The CD8+ TE cells mediated HA-1-specific DTH and produced interferon-γ. Suppression of these T E functions by TR cells was TGFβ, IL-10, and cytotoxic T lymphocyte-associated antigen 4 dependent. In addition, HA-1 microchimerism was detected in two recipients, primarily in the dendritic cell fraction of the PBMCs. This is the first demonstration of coexisting CD8 + memory TR and TE cells, both specific for the same HA-1 antigen, in the context of renal allograft tolerance.