Minor histocompatibility antigens (MiHAgs) cause slow-to-rapid organ transplant rejection by immunocompetent hosts and mild-to-severe graft-versus-host reactions in immunosuppressed hosts. MiHAgs are allelic forms of major histocompatibility complex (MHC) class I-restricted self-antigens recognized by cytotoxic T cells and usually are defined immunogenetically. Although structurally not identified as yet, it is assumed that MiHAgs are internal cell antigens that are processed and then presented by MHC class I proteins similar to viral antigens. To define a MiHAg both molecularly and functionally, we took advantage of the allelic difference of the structurally characterized intracellular myxovirus-resistance protein (Mx) and investigated its antigenicity. Skin grafts from congenic Mx+ mice carrying the functional Mx1 gene were rejected by mice lacking a functional Mx1 gene (Mx- mice). In parallel, cytotoxic MHC class I-restricted effector T cells specific for Mx protein and the H-2Kk antigen (but not for several other allelic H-2 antigens) were strongly induced in Mx- mice immunized with spleen cells from interferon-treated Mx+ mice. These data show that allelic forms of cell internal proteins presented by MHC class I may act as MiHAgs.