A variable number of tandem repeats (VNTR) polymorphism upstream of the insulin promoter is strongly associated with type 1 diabetes. The short class I alleles are predisposing and the long class III alleles are protective. As a possible mechanism for this effect, we previously reported a two- to threefold higher insulin transcription from class III than from class I chromosomes in thymus where insulin is expressed at low levels, presumably for the purpose of self-tolerance. In this article, we confirm this finding with independent methodology and report studies testing the hypothesis that class III alleles are associated with T-cell tolerance to (pro)insulin. Cytokine release in vitro after stimulation with 21 overlapping preproinsulin epitopes was assessed in blood mononuclear cells as well as naive and memory CD4+ T-cell subsets from 33 individuals with the high-risk DRB1*04, DQ8 haplotype (12 type 1 diabetic patients, 11 healthy control subjects, and 10 autoantibody-positive subjects). No significant differences between genotypes (24 I/I subjects versus 10 I/III or III/III subjects) were observed for gamma-interferon, tumor necrosis factor-alpha, or interleukin (IL)-4. By contrast, the I/III + III/III group showed a significant threefold higher IL-10 release in memory T-cells for whole proinsulin and the immunodominant region. Given that IL-10 is a marker of regulatory function, our data are consistent with the hypothesis that higher insulin levels in the thymus promote the formation of regulatory T-cells, a proposed explanation for the protective effect of the class III alleles.