Structural and functional studies of murine MHC class II I-A molecules have been limited by the low yield and instability of soluble, recombinant heterodimers. In the murine autoimmune diseases experimental autoimmune encephalomyelitis and collagen-induced arthritis, MHC class II molecules I-Au and I-Aq present peptides derived from myelin basic protein and type II collagen, respectively, to autoreactive T cells. To date, systems for the expression of these two I-A molecules in soluble form for use in structure-function relationship studies have not been reported. In the present study, we have expressed functional I-Au and I-Aq molecules using a baculovirus insect cell system. The chain pairing and stability of the molecules were increased by covalently linking the antigenic peptides to beta-chains and adding carboxyl-terminal leucine zippers. Peptide:I-Aq complex quantitatively formed an SDS-stable dimer, whereas peptide:I-Au formed undetectable amounts. However, the two complexes did not show any significant difference in their response to thermal denaturation as assessed by circular dichroism analyses. The autoantigen peptide:I-A complexes were highly active in stimulating cognate T cells to secrete IL-2 and inducing Ag-specific apoptosis of the T cells. Interestingly, the T cells were stimulated by these soluble molecules in the apparent absence of experimentally induced cross-linking of TCRs, indicating that they may have therapeutic potential in autoimmune disease models.