We have evaluated the effects of three potent immunosuppressive agents, cyclosporin A (CsA), FK506 and rapamycin, on the murine contact sensitivity(CS) reaction to the hapten trinitrochlorobenzene. Development of CS reaction requires participation of three distinct T cell subsets:αβ+, CD4+ T lymphocytes, which are the classical effector cell of the CS reaction, γδ+ T lymphocytes, andαβ+, double-negative (CD4- CD8-) T lymphocytes that express the B220 molecule and produce IL-4. We found that all three drugs inhibit the development of the CS reaction, but they affect different target cells. In fact, rapamycin and FK-506 block bothαβ+, CD4+ and γδ+ T lymphocytes, while CsA inhibits only the αβ+, CD4+ T lymphocyte. None of the three drugs exerted any inhibitory activity on theαβ+, double-negative (CD4- CD8-) T lymphocytes. Hapten-immune lymph node cells from mice treated in vivo with CsA or FK506 failed to proliferate and to produce IL-2 when re-exposed to the specific antigen in vitro. In contrast, immune lymph node cells from mice that had been treated in vivo with rapamycin gave optimal antigen-specific proliferation and IL-2 productionin vitro. The implications of these observations are discussed in relation to the use of these immunosuppressive agents for prevention of allograft rejection.