Type 1 diabetes is associated with autoimmune responses against insulin, with insulin autoantibodies (IAA) being a hallmark of the disease. Genetic susceptibility to type 1 diabetes is polygenic and includes the INS VNTR-IDDM2 locus, which has been shown to regulate the expression of insulin in the pancreas and thymus. In order to determine whether insulin autoimmunity could be attributed to a genetic susceptibility conferred by the INS-VNTR (IDDM2) locus, we studied the frequency of INS-VNTR alleles and the presence of IAA in 90 patients with new-onset type 1 diabetes. IAA were detected in 49 of 67 class I/I individuals and 19 of 23 class III/X patients, indicating that there is no association between the INS VNTR-IDDM2-susceptible allele and humoral autoimmunity against insulin. This finding does not support the hypothesis of an allele-specific tolerance induction that could determine susceptibility toward autoimmunity against the insulin protein and subsequently the development of type 1 diabetes.