The length of time after syngeneic bone marrow re-constitution when tolerance to alloantigens can be induced in adult mice during T cell differentiation from bone marrow cells was studied by exposing those T cells to (recipient × donor)F1spleen cells. Supralethally irradiated C3H/He Slc(C3H; H-2k) mice were reconstituted with 1×107syngeneic T cell-depleted bone marrow cells and then injected intravenously with 5×1107(C3H×C57BL/6[B6])F1(B6C3F1; H-2bxk) or (C3H × AKR/J[AKR])F1(AKC3F1; H-2kxk) spleen cells at various intervals.In the fully allogeneic combination of B6C3F1×C3H, EL-4 tumor originating from B6 was accepted, and survival of grafted B6 skin was significantly prolonged in the tolerant C3H mice treated with irradiation on day −1 followed by injection of syngeneic bone marrow cells on day 0 plus B6C3F1spleen cells on days 0, 5, or 10, in a tolerogen-specific manner. In the multiminor histocompatibility antigen—disparate combination of AKC3F1→C3H, AKR skin grafts were permanently accepted in the tolerant C3H mice treated with AKC3F1spleen cells on days 0, 5, 10, or 15. Immunological parameters, including cytotoxic T lymphocyte activity and delayed foot-pad reaction (DFR), were almost completely suppressed in C3H mice made tolerant of B6 or AKR antigens; A chimeric assay using a direct immu-nofluorescence method revealed that the tolerant C3H mice given B6C3F1spleen cells on day 0 were mixed-chimeric for at least 8 weeks after syngeneic bone marrow reconstitution, but not definitely chimeric thereafter. The C3H mice given AKC3F1spleen cells on day 0 were chimeric even 43 weeks after syngeneic bone marrow reconstitution, but the C3H mice given AKC3F1spleen cells on day 15 showed temporal chimerism that disappeared within 43 weeks. The untolerant mice were never detectably chimeric.These data suggest that the earlier the timing of the injection of F1spleen cells after syngeneic bone marrow reconstitution was, the more profound tolerance was induced. Moreover, the stronger the antigenic disparity between donor and recipient, the earlier the injection of F1spleen cells was required to induce tolerance.