While the effects of type II-interferon (IFN-gamma) on graft-versus-host disease (GVHD) and leukemia relapse are well studied, the effects of type I-interferon (type I-IFN, IFN-alpha/beta) remain unclear. We investigated this utilizing type I-IFN receptor-deficient mice and exogenous interferon-alpha (IFN-alpha administration in established models of GVHD and graft-versus-leukemia (GVL). Type I-IFN signaling in host tissue prevented severe colon-targeted GVHD in CD4-dependent models of GVHD directed towards either major histocompatibility antigens or multiple minor histocompatibility antigens. This protection was the result of suppression of donor CD4+ T cell proliferation and differentiation. Studies in chimeric recipients demonstrated this was due to type I-IFN signaling in hematopoietic tissue. Consistent with this, the administration of IFN-alpha during conditioning inhibited donor CD4+ proliferation and differentiation. In contrast, CD8-dependent GVHD and GVL effects were enhanced when type I-IFN signaling was intact in the host or donor respectively. This reflected the ability of type I-IFN to both sensitize host target tissue/leukemia to cell mediated cytotoxicity and augment donor CTL function. These data confirm that type I-IFN plays an important role in defining the balance of GVHD and GVL responses and suggests that administration of the cytokine after BMT could be studied prospectively in patients at high risk of relapse.