In previous studies, we demonstrated that intestinal epithelial cells of the mouse small intestine could present exogenous antigen to specific CD4+ T cell hybridomas. We now report on the ability of normal enterocytes to present the self superantigen Mls1a. Enterocytes from Mls1a but not from Mls1b strains stimulated interleukin-2 production through a V beta 6+ T cell hybridoma specific for Mls1a determinants. Antibody inhibition experiments showed that enterocytes presented Mls determinants via a major histocompatibility complex class II-dependent mechanism. Furthermore, the ability of enterocytes to activate V beta 6+ Mls1a-specific T cells was inhibited by monoclonal antibodies against the Orf protein encoded by an Mtv-7 provirus which is associated with Mls1a expression. These findings provide evidence for the first time that Mls determinants are expressed on normal enterocytes and support the theory of a possible role of these cells in extrathymic selection of T cell receptor V beta repertoire of intraepithelial T lymphocytes.