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In this report we demonstrate that the ion pair Arg-80α and Asp-57β, located in the peptide-binding site of nearly all class II major histocompatibility complex (MHC) proteins, is important for surface expression and function of the murine class II heterodimer I-Ad. Charge reversal at either of these two residues by site-directed mutagenesis generated mutant class II molecules that failed to appear at the cell surface. This defect in surface expression was partially reversed when the invariant chain was present or when the mutants were paired with the corresponding charge-reversed variant of the opposite chain. Surprisingly, surface expression was restored when cells expressing the single-site mutants were cultured at reduced temperature. In addition, the substitution of Asp-57β with residues found in alleles of class II molecules associated with diabetes resulted in heterodimers that were inefficiently expressed at the cell surface and presented foreign peptide poorly. Together, these results demonstrate that the formation of a salt-bridge between Arg-80α and Asp-57β is required for efficient surface expression of class II MHC molecules, therefore representing an important step in the assembly and transport of functional class II heterodimers to the cell surface.