Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post-BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD-inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.