Graded doses of Cyclophosphamide (Cy) or 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) were given to CD2F1 or C57Bl/6 mice. One, 45 or 60 days later the animals were tested for allograft responses, competence of producing cytotoxic lymphocytes in vitro and lethal graft-versus-host disease (GVHD) in vivo, delayed-type hypersensitivity (DTH) and humoral antibody responses against sheep red blood cells (SRBC). Both agents produced strong inhibitory effects, except for DTH, when given 1 day before the antigenic stimulus. However immunodepression lasted for at least 60 days after DTIC, whereas relatively rapid recovery of immune responsiveness was detected in mice treated with Cy. When Cy or DTIC were given to allogeneic donor mice 1 day before spleen cell transfer, immunodepressed recipients did not undergo GVHD. However when drugs were administered to recipient mice inoculated with allogeneic spleen cells, lethal GVHD occurred when Cy but not DTIC was given to the hosts. DTH responses were potentiated by Cy when the drug was given 1 day before sensitization. In contrast hypersensitivity reactions were not affected by DTIC treatment. It was concluded that DTIC is a potent and long-lasting immunodepressive agent, capable of affecting various T-cell subpopulations and possibly B lymphocytes in mice. Since the drug inhibits immune response when given before the antigenic stimulation, it was suggested that DTIC acts through a mechanism similar to that of alkylating non phase-specific agents.