We examined the mechanisms involved in interleukin (IL)-12-mediated suppression of cellular immunity in mice using allogeneic mixed leukocyte reaction (MLR) stimulated by dendritic cells (DCs) in vitro and compared the effect of IL-12 on MLR in mice and humans. Although IL-12 stimulated human MLR, the addition of IL-12 or interferon-γ (IFN-γ) resulted in a dose-dependent suppression of MLR in mice. The treatment with N G-monomethyl-L-arginine (L-NMMA) completely abrogated IL-12- and IFN-γ-mediated suppression of MLR in mice. Furthermore, IL-12 enhanced the alloreactive cytolytic T lymphocyte (CTL) induction in human MLR, whereas the addition of LNMMA was required to generate alloreactive CTLs in the presence of IL-12 in mice. Nitric oxide (NO) was detected only in mouse MLR. Murine DCs could produce NO, but neither human CD34+ cell- nor monocyte-derived DCs produced a detectable amount of NO. These results suggest that NO produced by DCs might play an important role in IL-12-mediated immune suppression in mice but not in humans.