Abstract To stimulate T cells, antigenic peptides must not only bind the presenting MHC molecule, but must form a complex of sufficient stability to interact with the T cell receptor (TCR). In a murine autoimmune gastritis model based on the transgenic expression of MHC-II restricted T cell receptors (TCR) directed against peptides from the alpha chain of the gastric parietal cell proton pump, the H/K ATPase, previous studies mapped residues 630-641 (PITAKAIAASVG), and residues 889-900 (PLLCVGLRPQWE) as the epitopes recognized by TXA23 and TXA51 transgenic T cells respectively. We studied the dissociation kinetics of peptides with various amino acid substitutions (including alanine-substitution at each non-alanine position). Binding to IAd was compared to T cell stimulatory activity of the same peptides in MHC-peptide complexes. Dissociation half lives at pH 5.3 (similar to endosomes) varied from ~20 hours to over 600 hrs. Peptide/MHC stability is greatest at neutral pH, where a nonstimulatory peptide (CLIP) and a highly stimulatory peptide (GHNLAVGLRPQWHE) had half lives approximately tenfold longer than at pH5.3. We find that a half-life of >20 hours at pH 5.3 is sufficient for a peptide with the necessary epitopic residues to retain immunogenicity. These results can be explained in the context of the X-ray structure of the IAd/PLLCVGLRPQWE complex. Supported by the NIAID intramural research program.