The cellular basis of the transplantation tolerance in a model system of BALB/c (Mls-1b) mice rendered cyclophosphamide (CP)-induced tolerant to DBA/2 (Mls-1a) skin allograft was investigated by assessing V beta 6+ T cells. From our results, three major mechanisms that are essential to the CP-induced skin allograft tolerance were sequentially elucidated. The first mechanism was destruction of donor-Ag-stimulated T cells in the periphery by CP treatment. The second mechanism was intrathymic clonal deletion of donor-reactive T cells, such as V beta 6+ T cells, correlating strongly with intrathymic mixed chimerism. The clonal deletion, however, was not always essential for the maintenance of the skin allografts, because DBA/2 skin survived even after the clonal deletion terminated and V beta 6+ T cells reappeared in the periphery of the recipient BALB/c mice. The third mechanism was generation of tolerogen-specific suppressor T cells, especially in the late stage of the tolerance. In contrast, the clonal anergy that is evidenced by the specific suppression of mixed lymphocyte reaction in the recipient BALB/c mice after injecting with DBA/2 spleen cells alone was not considered as a significant mechanism in prolonging skin allograft survival because such anergic mice showed accelerated rejection of the skin allografts. These results may suggest practical hierarchy of the mechanisms of CP-induced allograft tolerance.