I-Ad molecules harboring single amino acid changes in the conserved 80-82 region of the beta-chain show altered trafficking in invariant chain (Ii)-negative cell lines. Since residues beta81 and beta82 form hydrogen bonds with the backbone of bound peptide, alterations in this region may result in distinct MHC class II conformers that are targeted aberrantly. We examined the assembly and peptide binding properties of the mutant I-Ad molecules generated by in vitro translation. Indeed, loss of a single hydrogen bond at beta81, or of two hydrogen bonds at beta82, is sufficient to render I-Ad incapable of stable interaction with CLIP and other antigenic peptides, despite normal assembly with intact invariant chain. These results suggest that stable interaction of MHC class II molecules with peptide requires the integrity of the H-bond network between residues in the MHC class II alpha-helices and bound peptide, and that conformational features revealed by stable peptide binding are critical for MHC class II intracellular transport.