Successful active immunization against cancer requires induction of immunity against self or mutated self Ags. However, immunization against self Ags is difficult. Xenogeneic immunization with orthologous Ags induces cancer immunity. The present study evaluated the basis for immunity induced by active immunization against a melanoma differentiation Ag, gp100. Tumor rejection of melanoma was assessed after immunization with human gp100 (hgp100) DNA compared with mouse gp100 (mgp100). C57BL/6 mice immunized with xenogeneic full-length hgp100 DNA were protected against syngeneic melanoma challenge. In contrast, mice immunized with hgp100 DNA and given i.p. tolerizing doses of the hgp100 Db-restricted peptide, hgp10025-33, were incapable of rejecting tumors. Furthermore, mice immunized with DNA constructs of hgp100 in which the hgp10025-27 epitope was substituted with the weaker Db-binding epitope from mgp100 (mgp10025-27) or a mutated epitope unable to bind Db did not reject B16 melanoma. Mice immunized with a minigene construct of hgp10025-33 rejected B16 melanoma, whereas mice immunized with the mgp10025-33 minigene did not develop protective tumor immunity. In this model of xenogeneic DNA immunization, the presence of an hgp100 heteroclitic epitope with a higher affinity for MHC created by three amino acid (25 to 27) substitutions at predicted minor anchor residues was necessary and sufficient to induce protective tumor immunity in H-2b mice with melanoma.