Type 1 diabetes is a T-cell-mediated autoimmune disease in which insufficient regulatory mechanisms are perceived to be involved in the pathogenesis. We used flow cytometry to analyze the proportion of CD4(+)CD25(high) regulatory T cells and natural killer T (NKT) cells in peripheral blood obtained from 25 children with newly diagnosed type 1 diabetes, 21 nondiabetic subjects positive for two or more diabetes-associated autoantibodies, and from 39 autoantibody-negative age- and HLA-matched control subjects. CD4(+)CD25(high) T cells were also stained for additional markers HLA-DR, CD69, and CD62L. As NKT cell markers, we used CD161, V beta 11, and V alpha 24. The frequency of CD4(+)CD25(high)HLA-DR(-) T cells was significantly higher in multiple autoantibody-positive children than in controls (P = 0.021). We also detected a significantly higher level of CD4(+)CD25(high)HLA-DR(-) and CD4(+)CD25(high)CD69(-) T cells among children expressing three to four autoantibodies when compared to the controls (P = 0.004 and P = 0.048, respectively). The proportions of CD161(+)V beta 11(+) or V alpha 24(+)V beta 11(+) NKT cells were similar in all three groups of children studied. Interestingly, children with only two autoantibodies had a higher level of CD161(+)V beta 11(+) NKT cells than the controls (P = 0.002). Our data might be interpreted as indicative of an intensified regulatory response of regulatory T cells and NKT cells during the preclinical phase of the disease.