Tolerance to a vascularized allograft can be induced in adult animals by pregraft donor-specific blood transfusion (DST). Mechanisms underlying this effect appear to depend on unresponsiveness of alloreactive T-helper cells. In this study, we examined the roles of DST and cellular components of the allograft that are important in inducing T-cell unresponsiveness in a rat model. DST alone did not tolerize alloreactive recipient T-helper cells, but the combination of DST and heart allograft induced profound inhibition of the antidonor proliferative response in spleen but not in lymph node cells. When heart allografts were depleted of passenger leukocytes by pretreating the donor with cyclophosphamide or by parking the graft for 2 months in a tolerant recipient, tolerance induction in DST-treated recipients was abrogated. Tolerance could then be restored in a majority of DST-treated recipients of passenger leukocytes depleted grafts by injecting them at the time of grafting with donor, but not third-party, dendritic cells. This indicates that graft passenger leukocytes, most likely dendritic cells, are required for DST-induced allograft tolerance.