Many major histocompatibility complex (MHC) polymorphisms originate from ancient structures that predate speciation. As a consequence, members of the Mhc-DRB1*03 allelic lineage are not only present in humans but in chimpanzees and rhesus macaques as well. This emphasizes that Mhc-DRB1*03 members must have been present in a common ancestor of these primate species that lived about 30 million years ago. Due to the accumulation of genetic variation, however, alleles of the Mhc-DRB1*03 lineage exhibit species-unique sequences. To investigate the biological importance of such conservation and variation, we have studied both the binding and antigen presentation capacity of various trans-species Mhc-DRB1*03 lineage members. Here we show that p3-13 of the 65-kD heat-shock protein (hsp65) of Mycobacterium leprae and M. tuberculosis binds not only to HLA-DR17(3) but also to some chimpanzee and rhesus macaque class II-positive cells. Comparison of the corresponding human, chimpanzee, and rhesus macaque Mhc-DRB1*03 lineage members revealed the presence of uniquely shared amino acid residues, at positions 9-13 and 26-31, of the antigen-binding site that are critical for p3-13 binding. In addition it is shown that several nonhuman primate antigen-presenting cells that bind p3-13 can activate HLA-DR17-restricted T cells. Certain amino acid replacements, however, in Mhc-DRB1*03 lineage members did not influence peptide binding or T cell recognition. Therefore, these studies demonstrate that some polymorphic amino acid residues (motifs) within the antigen-binding site of MHC class II molecules that are crucial for peptide binding and recognition by the T cell receptor have been conserved for over 30 million years.