The human major histocompatibility complex includes approximately 14 class II HLA genes within the HLA-D region, most of which exist in multiple allelic forms. One of these genes, the DQ3.2 beta gene, accounts for the well-documented association of HLA-DR4 with insulin-dependent diabetes mellitus and is the single allele most highly correlated with this disease. We analyzed the amino acid substitutions that lead to the structural differences distinguishing DQ3.2 beta from its nondiabetogenic, but closely related allele, DQ3.1 beta. Site-directed mutagenesis of the DQ3.2 beta gene was used to convert key nucleotides into DQ3.1 beta codons. Subsequent expression studies of these mutated DQ3.2 beta clones using retroviral vectors defined amino acid 45 as critical for generating serologic epitopes characterizing the DQw3.1 beta and DQw3.2 beta molecules.