The role of peripheral hemopoietic chimerism in the induction and maintenance of donor-specific tolerance was investigated by our tolerance-inducing method using cyclophosphamide (CP). As has been previously reported, CP injection at a dose of 200 mg/kg to C3H (Thy-1.2, Mls-1b) mice 2 days after priming with 10(8) viable AKR (Thy-1.1, Mls-1a) spleen cells (SC) resulted in both establishment of mixed chimerism and selective elimination of V beta 6+CD4+ T cells in the periphery. When, instead of viable SC, 1300 rad irradiated 10(8) AKR SC were used for priming to C3H mice, CP treatment 2 days after the priming also caused significant but, as compared with priming with nonirradiated viable cells, incomplete elimination of V beta 6+ T cells in the periphery. In these mice, no hemopoietic chimerism was found. In parallel with this incomplete elimination of peripheral V beta 6+ T cells, LN cells of these mice showed reduced but considerable response to AKR SC. However, once hemopoietic chimerism was introduced to these incompletely tolerant mice by an injection with donor-type viable [AKR x C3H]F1 SC 2 days after CP-treatment, LN cells from these newly established chimeras, irrespective of presence or absence of the thymus, became completely nonresponsive to AKR while preserving normal response to BALB/c (third party). This state of nonresponsiveness was accompanied by clonal elimination of the remaining V beta 6+ T cells in the periphery. These results indicate that peripheral chimerism promoted profound tolerance to donor-Mls Ag specifically. Furthermore, from experiments of skin grafting, we demonstrated that tolerance to minor histocompatibility Ag was also achieved in the presence of peripheral hemopoietic chimerism.