Polymorphism is known to exist within the HLA-DQ alpha locus in the human major histocompatibility complex, although such polymorphism may be "silent" in standard HLA typing. However, DQ alpha polymorphism may be functionally significant, either through DQ alpha epitopes functioning directly in the immune response or by affecting tertiary conformation of Ia molecules through differential alpha/beta pairing. We have previously defined a particular DQ alpha polymorphism through reactivity with a monoclonal antibody and restriction fragment length polymorphism pattern. We now characterize this DQ alpha polymorphism through two-dimensional gel electrophoretic analysis and identify a subset of DQ alpha molecules with unique characteristics. Investigation of these allelic variants using synthetic oligonucleotide probe analysis of genomic DNA suggests a localization of the DNA region encoding the DQ alpha 5 epitope and suggests possible evolutionary mechanisms accounting for these unique patterns.