For a broader view of the role of H-2M as an accessory molecule in antigen presentation, we investigated the degree to which different MHC class II isotypes and alleles depend on H-2M to function in vivo. We generated H-2M-deficient animals expressing Ek/b or Ak molecules in addition to the Ab molecules already present in the mutant strain, and compared the ability of the different MHC class II molecules to present antigen at the cell surface for recognition by T cells, and contribute to positive selection of CD4+ T cells in the thymus. Biochemical analyses were performed to assess MHC class II maturation, and to determine the peptide content of the molecules. In the absence of H-2M, Ek/b molecules contained a more heterogeneous set of class II-associated invariant chain peptides (CLIP) than Ab did, which, unlike Ab-CLIP complexes, were not SDS-stable. Unlike Ab molecules, both Ek/b and Ak efficiently presented exogenously added peptides to T cells in the absence of H-2M. In addition, epitopes from some proteins, especially those known to be invariant chain independent, were presented by Ak molecules in the mutant animals. To our surprise, expression of Ek/b overcame the positive selection defect observed in H-2M-deficient mice expressing Ab alone. In contrast, Ak expression did not augment positive selection of CD4+ T cells in the mutant animals. Some of these findings in vivo contrast significantly with findings from in vitro studies on murine MHC class II molecules in human DM-deficient cell lines.