Background. One of the complexities of solid organ allograft rejection is the inherent diversity of the specific T cell antigenic epitopes that participate in this response, including the role of direct alloantigen recognition and indirect recognition of donor-derived peptides in recipient antigen-presenting cells. To probe the role of distinct T cell receptor (TCR) avidity differences and the role of cytokine expression patterns of different effector T cells that may participate in allograft rejections, we have identified a dominant allopeptide derived from the H-2K(d) molecule, recognized by H-2b CD4 T cells in the context of syngeneic I-Ab. Methods. To identify a stimulatory peptide derived from the H-2K(d) molecule, a panel of synthetic overlapping peptides was screened for immunogenicity and a panel of T cell clones established. These clones were characterized for TCR Vβ usage by mAb staining and/or reverse transcribed-polymerase chain reaction analysis, peptide dose sensitivity as a marker of TCR avidity, cytokine expression phenotype in vitro, and their ability to mediate rejection of a vascularized cardiac allograft after adoptive transfer to immunodeficient mice. Results. The H-2K(d)54-68 peptide was identified as a dominant stimulatory peptide by the ability of T cells from C57BL/6 (H-2b) mice primed by a combination of allogeneic spleen cell injection and mixed peptide immunization to mount an in vitro proliferative response and interferon-γ production by peptide stimulation. Furthermore, direct immunization with synthetic H-2K(d)54-68 peptide of normal C57BL/6 mice resulted in accelerated rejection of both skin and cardiac allografts from B10.D2 (H-2(d)) mice, but not 3rd party B10.BR (H-2(k)) grafts. A panel of 15 distinct CD4+ clones specific for H-2K(d)54-68 peptide were established and shown to utilize a variety of TCR Vβ and different apparent TCR avidities to H-2K(d)54-68 peptide when stimulated in vitro. To characterize these clones further, two clones were chosen based on the difference of avidity to H-2K(d)54-68 peptide. The cytokine expression pattern was determined and indirect alloantigen specificity confirmed by analysis of responses to purified peptide and B10.D2 spleen cells using normal H-2b and I-Aβ chain knockout mice as APC donors. Both of these T cell clones were able to mediate rejection of B10.D2, but not B10.BR hearts, in immunodeficient mice, but the morphological pattern of T cell infiltration was distinct. Conclusions. These results demonstrate the potential importance of fine dissection of the alloantigeneic response to solid organ transplants and provide unique insights into the role of TCR avidity and cytokine expression patterns in different morphological patterns of transplant rejection.