The DR3-restricted peptide, MT 65 kDa 3-13, was used to develop a DR3-specific binding assay. The binding activity detected was strictly pH-dependent, in that it was optimal in the pH 4 to 5 range, and no activity was detected at neutral pH. By means of affinity chromatography purifications and the use of DR3-transfected fibroblasts, it was shown that no cross-reactivity exists at the level of DR52a molecules, thus allowing use of only partially purified DR3/DR52a mixtures in high throughput binding assays. The immunologic relevance of the assay established was also verified by examining the correlation between DR3 restriction and binding for a panel of DR-restricted peptides. When the structural requirements for peptide DR3 interactions were further examined by using panels of analogues of two different epitopes (Myo 132-151 and TT 830-843), it was found that DR3 molecules recognized a peptide motif distinct from the one recognized by the other major DR beta 1 alleles.