Mouse mammary tumor virus (MMTV) is a retrovirus encoding a superantigen that is recognized in association with major histocompatibility complex class II by the variable region of the beta chain (V) of the T-cell receptor. The C-terminal 30 to 40 amino acids of the superantigen of different MMTVs display high sequence variability that correlates with the recognition of particular T-cell receptor V chains. Interestingly, MMTV(SIM) and mtv-8 superantigens are highly homologous but have nonoverlapping T-cell receptor V specificities. To determine the importance of these few differences for specific V interaction, we studied superantigen responses in mice to chimeric and mutant MMTV(SIM) and mtv-8 superantigens expressed by recombinant vaccinia viruses. We show that only a few changes (two to six residues) within the C terminus are necessary to modify superantigen recognition by specific Vs. Thus, the introduction of the MMTV(SIM) residues 314-315 into the mtv-8 superantigen greatly decreased its V12 reactivity without gain of MMTV(SIM)-specific function. The introduction of MMTV(SIM)-specific residues 289 to 295, however, induced a recognition pattern that was a mixture of MMTV(SIM)- and mtv-8-specific V reactivities: both weak MMTV(SIM)-specific V4 and full mtv-8-specific V11 recognition were observed while V12 interaction was lost. The combination of the two MMTV(SIM)-specific regions in the mtv-8 superantigen established normal MMTV(SIM)-specific V4 reactivity and completely abolished mtv-8-specific V5, -11, and -12 interactions. These new functional superantigens with mixed V recognition patterns allowed us to precisely delineate sites relevant for molecular interactions between the SIM or mtv-8 superantigen and the T-cell receptor V domain within the 30 C-terminal residues of the viral superantigen.