To examine the nature of HLA-linked genetic susceptibility to insulin-dependent diabetes mellitus (IDDM), we compared HLA class II gene sequences from IDDM patients and control individuals. Genomic libraries were constructed from two siblings with IDDM, typed serologically as DR3,w6 and DR3,4. These libraries represent the HLA haplotypes (DR3, DR4) most frequently associated with IDDM, as well as one haplotype found less often. Individual genomic clones were identified and assigned to specific loci and haplotypes. The nucleotide sequence was then determined from the variable second exon from the HLA-DQ alpha, DQ beta, and DR beta genes from all three haplotypes. Sequence variation within the DQ alpha genes could not be correlated with the disease. For all three haplotypes, the DQ alpha sequence from the IDDM patient was identical to the DR-matched control sequence. Similarly, for the DR3 haplotype, the DQ beta sequences matched all control DR3 alleles. The DQ beta sequence from the DR4 haplotype was identical to the predominant DR4 allele (DQ beta 3.2) but differed at four amino acid residues from the other major DR4 DQ beta sequence (DQ beta 3.1) found rarely among IDDM patients. Sequence analysis of the DQ beta gene from the DRw6 haplotype revealed a new allele that differed from the DQ beta allele from a control DR6 allele at two residues. The DR beta genes from these three haplotypes also did not show any sequence features uniquely associated with IDDM, although the frequency of certain allelic variants in all three of these haplotypes may be altered in the IDDM population. A particular group of amino acids was found to be shared between the DR beta-1 alleles from the DR4 and DRw6 haplotypes and may be involved in genetic susceptibility to IDDM.