To investigate the role of the indirect alloantigen pathway in transplant rejection, we established T cell clones specific for an allogeneic class I peptide. A series of overlapping 15-mer peptides corresponding to the variable regions of H-2Kd molecule were synthesized and screened by measuring T cell proliferation and IFN-γ production. Responder cells in this assay were draining lymph node cells from C57BL/6 (H-2b) mice primed by the combination of i.p. injection of B10.D2 (H-2d) spleen cells and a mixture of peptides in adjuvant. Several peptides demonstrated activity in this assay and a single one (H-2Kd 75-89) was chosen for further study. Direct immunization of C57BL/6 mice with this peptide caused subsequent accelerated rejection of H-2d skin or cardiac allografts, but not H-2k allografts. Several CD4+ T cell clones were generated via direct immunization with the synthetic peptide, which showed variability in the cytokine expression phenotype, but were all specific for the I-Ab/H-2Kd75-89 epitope. Two clones with differing antigen receptor avidity (peptide dose-response in vitro) and differing cytokine patterns could mediate cardiac allograft rejection of H-2d, but not H-2k, hearts in B6-SCID hosts that received the allograft 2 weeks prior to T cell clone transfer. Although complete rejection occurred within a week after in vivo transfer with both of these clones, histopathological analysis demonstrated differences in the apparent mechanism of rejection. These findings indicate that H-2Kd 75-89 peptide binds to I-Ab, stimulates a diverse population of T cells, and that these cells can find sufficient amounts of the indirect peptide epitope within a cardiac allograft to mediate rejection.