CD4+CD25+ T cells (Tregs) play a critical role in maintaining dominant peripheral tolerance, and pathogenic autoreactive T cells may be frequent in the CD25-negative fraction of peripheral blood mononuclear cells (PBMCs) from patients with autoimmune disease. We therefore investigated whether T cell autoimmune responses to recombinant GAD65 can be detected by the use of ELISPOT assay in the CD25-negative fraction of PMBCs from Japanese type 1 diabetes (T1D) patients. The frequency of CD4+CD25+ T cells was not different among patients with newly developed T1D, established T1D, and healthy controls. The CD25 positive cell–depleted fraction was obtained by negative selection with antihuman CD25 magnetic beads, reducing the number of CD4+CD25+ T cells from 4–5% to less than 1%. In whole PBMC fraction, there was a significant elevation of IFN-γ spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN-γ spots from patients with established T1D was not significant. In the CD25-negative fraction, unlike whole PBMCs, we observed the significant IFN-γ spots to GAD65 in the fraction from patients with established T1D (P < 0.05), but not in those with recently diagnosed disease. The phenomena were not observed for IL-4 spots. Our data suggest a possible role of Tregs maintaining dominant peripheral tolerance in T1D and application of further improved T cell assay detecting autoimmunity even in established T1D.