SUMMARYThe development of acute graft-versus-host (GVH) disease in (C57BL/Rij X CBA/Rij)F, hybrid mice inoculated with C57BL/Rij spleen cells is associated with a specific antihost delayed-type hypersensitivity (DTH) in the lymphoid organs of these mice. In this paper the role of proliferation in the development of this DTH response was studied in relation to the cellular changes in spleen and lymph nodes during GVH disease.Blocking of DNA synthesis of the C57BL/Rij spleen cells by incubation with 25 /μg of mitomycin C per ml before inoculation into the irradiated recipients did not prevent the antihost DTH responsiveness in the spleen during the first 2 days, and in the lymph nodes at 3 days after reconstitution. Thereafter, the DTH responsiveness was greatly decreased by the mitomycin C treatment. Blocking of both DNA and RNA and a great part of protein synthesis by pretreatment with 100 μg of mitomycin C per ml could completely prevent the development of DTH responsiveness in the recipient mice.Elimination of the DNA synthetizing cells from the recipient's spleen and lymph nodes by tritiated thymidine ([3H]TdR) suicide in vitro revealed that the appearance of antihost DTH responsiveness in both organs is associated with a local proliferation of the reactive T cells. In both the spleen and lymph nodes, elimination of the DNA synthetizing cells at the moment of peak DTH reactivity reduced the DTH response to about 50% of the normal value. This decrease was larger before reaching peak DTH reactivity, whereas thereafter [3H]TdR could no longer affect the height of the antihost DTH response. These results suggest that full development of specific antihost DTH responsiveness during an acute GVH reaction is dependent on proliferation of the reactive T cells.