The HLA association with insulin-dependent diabetes mellitus is highest among individuals heterozygous for DR3 and DR4. To investigate potential mechanisms to account for this association, we performed two-dimensional gel-electrophoretic analysis of HLA molecules from DR3/4 heterozygous patients. These studies demonstrated hybrid molecular dimers corresponding to products of HLA-DQ genes linked to DR3 and DR4, i.e., the DQw2 and DQw3 genes, respectively. Two types of OQ molecules were found: immunoprecipitation by DQw3-specific monoclonal antibody 17.15 identified a DQw3 β-chain associating with a DQw3 α-chain and a DQw3 β-chain associating with a DQw2 α-chain. The identity of α- and β-chains comprising these hybrid molecules was confirmed by HPLC peptide-map analysis. Several characteristic peptide peaks identified both DQw2 and DQw3 α-chains associated with DQw3 β-chains. The formation of such DQα(DQw2)- Dqβ(DQw3) dimers potentially contributes a direct molecular mechanism for HLA-associated contributions to disease in DR3/DR4 heterozygotes.