The phenotype of T cells that initiate graft-vs-host disease (GVHD) in response to minor histocompatibility antigens (minor HA) was determined in three H-2 compatible strain combinations by using negative selection with monoclonal antibodies to Lyt-2 and L3T4 antigens to test the hypothesis that Lyt-2-positive T cells alone initiate GVHD. The phenotype of T cells required to initiate GVHD was different in each of the three strain combinations studied. Both Lyt-2+ and L3T4+ LP spleen cells were necessary to cause lethal GVHD in C57BL/6 recipients. In the reciprocal transplant, Lyt-2+, but not L3T4+ C57BL/6 spleen cells were sufficient to initiate GVHD in LP recipients. In contrast, L3T4+, but not Lyt-2+ B10.D2 spleen cells were found to initiate GVHD in BALB/c recipients. The optimal response to minor HA requires both Lyt-2+ and L3T4+ T cells because a mixture of the two subsets of spleen cells resulted in a more severe form of GVHD than either subset alone in all three strain combinations studied. This study demonstrates that L3T4+ cells participate in the initiation of GVHD in response to minor HA. The dominant T cell subset that initiates GVHD varies with the specific strain combination tested. The specific minor HA expressed in the transplant recipient, the H-2 type, and possibly non-major histocompatibility complex immune response genes of the donor strain appear to determine the phenotype of the initiator T cells.