Mammary tumor viruses (MMTVs) as well as their endogenous counterparts encode superantigens which react with T cells expressing particular T cell receptor V beta chains. Several lines of evidence indicated that MHC class II is required for the functional presentation of these superantigens. Here we provide direct proof that the function of superantigens is abrogated in the absence of MHC class II expression. No deletion of Mls-1-reactive T cells was observed in MHC class II-deficient mice, and splenocytes from these animals did not stimulate Mls-1-reactive T cell hybrids in vitro. Furthermore, the viral spread in MHC class II-deficient mice, maternally infected with MMTV(C3H), was severely reduced. While initial infection in the gut-associated lymphocytes was comparable between MHC class II-deficient and normal mice, the level of infection in the spleen and the mammary tissue was much lower in the deficient animals. Quantitation of proviral DNA in spleen revealed a direct correlation between the magnitude of superantigen stimulation and degree of infection. These experiments document the direct effect of superantigen stimulation on viral amplification.