T cell recognition of xenoantigens is likely to play a key role in rejection of xenografts surviving hyperacute and delayed xenograft rejection, but the mechanisms of how this might occur are unknown. We used synthetic rat class II MHC peptides to study the role of the indirect pathway, where processed xenogeneic MHC antigens are presented in the context of self MHC, in a concordant xenograft rejection model in vivo. Mice of four different strains, BALB/c, B1O.A, CBA/ca, and C57BL/6j, were immunized with a mixture of rat class II MHC 25-mer xenopeptides representing the full-length sequence of the beta chain hypervariable domain of either RT1.Du (DR and I-E like) or RT1.Bu (DQ and I-A like) of the Wistar-Furth (WF) (RT1u) rat. Draining lymph node cells were capable of recognizing and proliferating to specific class II xeno-MHC peptides. The immunogenicity of the different peptides varied with the responder mouse strain. Responder T cells were CD4+, and were inhibited by anti-I-A and anti-I-E antibodies. We then examined the proliferative response of T cells from B1O.A primed by WF skin or vascularized cardiac xenografts to the class II MHC xenopeptides, when presented by naive B1O.A splenic antigen-presenting cells. These T cells were capable of proliferating to the same xeno-MHC peptides shown to be immunogenic by immunization. These data confirm the occurrence of self-restricted T cell recognition of xeno-MHC peptides in xenograft rejection, and provide the rationale for further investigating the role of the indirect pathway of recognition in xenotransplantation.