Several genomic polymorphisms at the insulin(INS)gene and its flanking regions were analyzed in 197 unrelated Caucasian patients affected by insulin-depenent diabetes (IDDM) and 159 ethnically matched, normal controls ascertained from the South-Eastern United States. We found that the frequency of homozygotes for the common variant at the insulin gene was significantly increased in the diabetic population (RR = 2.0, p<0.005). However, the polymorphisms in the 5′and 3′regions flanking theINSwere not significantly associated with IDDM. These results suggest that the IDDM susceptibility locus on chromosome 11p is located within the region extending from the 5′VNTR to the 3′end of theINSgene. We determined theHLA-DQB1genotypes by denaturing gradient gel electrophoresis (DGGE) and/or sequence-specific primers (SSP) techniques to assess the possible interactions betweenINSand HLA.DQB1*0302had the strongest predisposing effect on IDDM susceptibility (RR = 9.3) andDQB1*0602the strongest protective effect (RR = 0.02). However, a significant predisposing effect ofDQB1*020/could be demonstrated only after removal of the effects ofDQB1*0302andDQB1*0602.Analyses of the genotypes revealed that all genotypes containing 0602 were protective and that the heterozygous genotype 0201/0302 and homozygous genotype 0302/0302 confer the highest risk (RR = 20.9 and 12.9 respectively). However, heterozygous genotypes 0302/X (X excludes 0201,0302 and 0602) have a significantly lower predisposing risk. Similarly, there is heterogeneity in risk between predisposing 0201/0201 homozygous individuals and protective 0201/X individuals. When subjects were stratified by HLA genotypes, the relative risks conferred byINSdid not vary, thus suggesting that the susceptibility effects conferred by HLA andINSare additive rather than interactive.