Abnormalities in insulin regulation are central to the pathogenesis of type 2 diabetes. We assessed variation in the insulin gene variable number tandem repeat (INS VNTR) minisatellite (using the -23Hph1 A/T single nucleotide polymorphism) as a risk factor for 92 cases of incident type 2 diabetes in 883 unrelated Framingham Heart Study (FHS) subjects and in a separate sample of 698 members of 282 FHS nuclear families with 62 diabetes cases. In the unrelated sample, the -23Hph1 TT genotype frequency was 8.0% and was associated with a diabetes hazard ratio of 1.89 [95% confidence interval (CI), 1.01-3.52; P = 0.045] compared with the AA genotype using diabetes age of onset as the time failure variable in a proportional hazards model adjusted for age, offspring sex, body mass index, parental diabetes, and sex by parental diabetes interactions. In sex-stratified analyses, TT increased risk for diabetes in women (hazard ratio, 4.25; 95% CI, 1.76-10.3), but not men (hazard ratio, 1.01; 95% CI, 0.39-2.60). Using a family-based association test to assess transmission disequilibrium in the sample of related subjects, the age- and sex-adjusted z-score for diabetes associated with the T allele was 2.07 (P = 0.04), and a family-based association test using age of onset in a proportional hazards model was also statistically significant (P = 0.03), indicating that increased risk of diabetes was not attributable to population admixture. These data support the hypothesis that the INS VNTR is a genetic risk factor for type 2 diabetes, with the TT genotype accounting for about 6.6% of cases in the FHS population.