The stability of a specific complex between the peptide Ag representing residue 323-339 of OVA and the MHC class II protein, I-Ad, in a lipid bilayer was investigated as a function of pH and temperature. The complex is much more stable in a lipid bilayer than previously reported for detergent micelles. Measureable dissociation was detectable only after several hours at a pH below 5. The results show that a purified preparation of MHC class II molecules can sequentially bind, release, and rebind peptide, indicating that, in principle, MHC class II molecules could be used more than once for peptide binding. However, the time and pH required for peptide-MHC dissociation suggests that, in an Ag presenting cell, either a prolonged residence in an acidic compartment or other factors will be required for regeneration of the peptide binding site.