Superantigens (SAG) presented in the context of MHC class II proteins stimulate a strong proliferative response in T cells expressing particular TCR V beta genes. Although this T-cell recognition is not MHC restricted, a strong hierarchy is observed in the ability of various MHC class II molecules to present SAG. Mls-1, encoded by the Murine Mammary Tumor Virus (MMTV) Mtv-7 sag gene, is the prototype of endogenous SAG. In the present study, we have analyzed whether this retroviral gene product can be presented in the context of human HLA class II proteins to murine T cells. Positive results were obtained with the DR isotype and in in vitro, as well as in vivo T-cell stimulation assays. However, the various DR beta alleles, expressed in combination with an identical DR alpha chain, differed in their Mls-1 presenting capacity, indicating that the MHC class II beta-chain contains the primary contact site for Mls-1. Interestingly, the same pattern of TCR V beta restriction was seen in response to Mls-1 presented in the context of human and mouse class II, suggesting that the TCR V beta specificity is uniquely determined by the retroviral SAG. Furthermore, Mls-1 presented in the context of the DQw1 and DPw2 isotypes did not elicit a T-cell response. The results from this study form the basis for further analysis of the exact region in the class II beta-chain that interacts with Mls-1.