The role played by antigenic peptides bound to major histocompatibility complex (MHC) molecules is evaluated with H2-DMα^−/− mice. These mice have predominantly class II–associated invariant chain peptide (CLIP)-, not antigenic peptide–bound, MHC class II. H2-DMα^−/− donor heart grafts survived three times longer than wild-type grafts and slightly longer than I-A_β^b−^/− grafts. Proliferative T cell response was absent, and cytolytic response was reduced against the H2-DMα^−/− grafts in vivo. Residual cytolytic T cell and antibody responses against intact MHC class I lead to eventual rejection. Removal of both H2-DMα and β2-microglobulin (β2m) in cardiac grafts lead to greater (8–10 times) graft survival, whereas removal of β2m alone did not have any effect. These results demonstrate the significance of peptide rather than just allogeneic MHC, in eliciting graft rejection.