To study the role of thymic education on the development of the human T cell repertoire, SCID-hu mice were constructed with fetal liver and fetal thymus obtained from the same or two different donors. These animals were studied between 7 and 12 mo after transplantation, at which times all thymocytes and peripheral T cells were derived from stem cells of the fetal liver graft. Immunohistology of the thymus grafts demonstrated that thymic epithelial cells were of fetal thymus donor (FTD) origin. Dendritic cells and macrophages of fetal liver donor (FLD) origin were abundantly present in the medullary and cortico-medullary areas. Thymocytes of SCID-hu mice transplanted with liver and thymus of two different donors (FLDA/FTDB animals) were nonresponsive to Epstein-Barr virus-transformed B cell lines (B-LCL) established from both the FLDA and FTDB, but proliferated vigorously when stimulated with third-party allogeneic B-LCL. Mixing experiments showed that the nonresponsiveness to FTDB was not due to suppression. Limiting dilution analysis revealed that T cells reacting with the human histocompatibility leukocyte antigens (HLA) of the FLD were undetectable in the CD8+ T cell population and barely measurable in the CD4+ subset. On the other hand, CD4+ and CD8+ T cells reactive to the HLA antigens of the FTD were readily detectable. These results indicate that FLD-reactive cells were clonally deleted, whereas FTD-reactive cells were not. However, the frequencies of FTD-reactive T cells were consistently twofold lower than those of T cells specific for any third-party B-LCL. In addition, the cytotoxic activity and interleukin 2 production by FTD-specific T cells were lower compared with that of third-party-reactive T cell clones, suggesting that FTD-specific cells are anergic. These data demonstrate that T cells become tolerant to autologous and allogeneic HLA antigens expressed in the thymus via two different mechanisms: hematopoietic cells present in the thymus induce tolerance to "self"-antigens by clonal deletion, whereas thymic epithelial cells induce tolerance by clonal energy and possibly deletion of high affinity clones.