In H2-DM mutant mice, most MHC class II molecules are bound by a single peptide, CLIP, derived from the class II-associated invariant chain. Previous studies showed that H2-DM- cells are defective in presenting synthetic peptides to class II-restricted T cells. In sharp contrast, however, the same peptides elicited strong CD4+ T cell responses in H2-DM- animals. We now provide an explanation for this apparent discrepancy. Peptide-specific CD4+ T cells from wild-type mice were efficiently stimulated by H2-DM+, but not by H2-DM- cells pulsed with the cognate peptide. In sharp contrast, CD4+ T cells from mutant animals specific for the same MHC-peptide combination recognized peptide-pulsed H2-DM+ and H2-DM- cells equally well. In addition, unlike Ag-specific T cells from wild-type animals, the reactivities of peptide-specific T cells from mutant animals could not be efficiently blocked by Abs specific for the cognate MHC class II-peptide combination. We further demonstrated that the distinct reactivities of CD4+ T cells from H2-DM+ and H2-DM- mice are due to differences in thymic selection. Collectively, these findings indicate that the CD4+ T cell repertoires of H2-DM+ and H2-DM- mice are remarkably different.