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To determine the potential role of macrophages as regulators of cell-mediated immunity, the additive effect of rat peritoneal macrophages on the graft-versus-host reactivity of parental lymphoid cells was investigated. Local graft-versus-host reactivity of parental lymph node cells and thymus cells, as measured by the popliteal lymph node enlargement assay, was augmented greatly by the addition of an appropriate dose of macrophages. This augmenting effect was also confirmed his-tologically, by showing the higher degree of blastogenesis in the side supplemented with macrophages than that of the control side. Furthermore, macrophages from either the syngeneic or allogeneic genotype can also greatly augment the graft-versus-host reaction in both parental lymph node and thymus cell inoculation, indicating that the collaborative effect of macrophages may be exerted over histocompatibility barrier.In the experiments of parental thymus cell inoculation, the augmenting effect of macrophages was observed more greatly than that of lymph node cell inoculum. When cortisone-resistant thymus cells were injected into F1 hybrid rats, no augmenting effect of macrophages was observed. These findings indicate that a possible target for the macrophage action may be subclasses of peripheral T cells and cortisone-sensitive thymus cells from donor origin. Thus, during the graft-versus-host reaction in vivo, macrophages appeared to play a collaborative role in influencing the reactivity of parental T cells to alloantigen and also in the functional maturation of subclasses of thymus cells.