Mechanisms of cyclophosphamide (CP)-induced tolerance were studied. When AKR/J Sea (AKR: H-2k) mice were primed i.v. with 5 x 10(7) spleen cells plus 1 x 10(7) bone marrow cells from [C57BL/6 Slc (B6; H-2b) x C3H/He Slc (C3H; H-2k)]F1 (B6C3F1) mice and treated i.p. with 200 mg/kg CP 2 days later, the survival of C3H skin was moderately prolonged, but the survival of either B6 or B6C3F1 skin was not prolonged. By this treatment, however, mixed chimerism of B6C3F1 cells in the AKR mice was not established. When C3H cells were used as the tolerogen, a minimal degree of mixed chimerism associated with profound tolerance to C3H skin was established. Similar results were observed in various donor-recipient combinations. When C3H skin was grafted in the AKR mice 12 wk after the treatment with C3H cells and CP, or B6C3F1 cells and CP, survival of the grafted C3H skin was prolonged remarkably or moderately, respectively, although mixed chimerism was not detectable at the timing of grafting in either of the groups. In this late stage of tolerance, a strong level of tolerogen-specific suppressor cell activity was observed in those tolerant AKR mice. The suppressor activity was mainly attributable to T cells. These results suggest that the role of Ts cells in order to maintain skin tolerance is important in our CP-induced tolerance system, especially in the late stage of tolerance. Moreover, the generation of the Ts cells does not necessarily require the establishment of a long term mixed chimeric state.