The study investigates the peptides bound to HLA-DR alleles, focusing on their specificity and promiscuity. Using mass spectrometry and Edman microsequencing, over 200 unique peptide masses were identified from each of the five HLA-DR alleles (DR2, DR3, DR4, DR7, and DR8), ranging from 1,200 to 4,000 daltons. These peptides were derived from 66 different source proteins, with most (over 85%) originating from endogenous proteins that intersect the endocytic/class II pathway. The peptides were predominantly derived from major histocompatibility complex-related molecules, with a few derived from exogenous bovine serum proteins. Four sets of promiscuous self-peptides and 84 allele-specific peptide sets were identified. Binding experiments confirmed that these promiscuous peptides have high affinity for all HLA-DR alleles examined. The potential role of these endogenous self-peptides as immunomodulators of the cellular immune response is discussed. The study highlights the complexity and diversity of peptides bound to HLA-DR alleles, providing insights into antigen processing and peptide binding mechanisms.The study investigates the peptides bound to HLA-DR alleles, focusing on their specificity and promiscuity. Using mass spectrometry and Edman microsequencing, over 200 unique peptide masses were identified from each of the five HLA-DR alleles (DR2, DR3, DR4, DR7, and DR8), ranging from 1,200 to 4,000 daltons. These peptides were derived from 66 different source proteins, with most (over 85%) originating from endogenous proteins that intersect the endocytic/class II pathway. The peptides were predominantly derived from major histocompatibility complex-related molecules, with a few derived from exogenous bovine serum proteins. Four sets of promiscuous self-peptides and 84 allele-specific peptide sets were identified. Binding experiments confirmed that these promiscuous peptides have high affinity for all HLA-DR alleles examined. The potential role of these endogenous self-peptides as immunomodulators of the cellular immune response is discussed. The study highlights the complexity and diversity of peptides bound to HLA-DR alleles, providing insights into antigen processing and peptide binding mechanisms.