Insulin-specific immune responses appear early in preclinical type 1 diabetes (T1D), and bovine insulin in cow's milk-based infant formulas has been suggested to be of importance in induction of the primary response to insulin in humans. To characterize insulin-specific T-cell reactivity we studied T-cell responses to 10 insulin peptides derived from bovine (BI) and human insulin (HI) in 42 children with recently diagnosed T1D, 47 children with multiple autoantibodies and 111 autoantibody-negative control children with risk-associated HLA alleles. Proliferation responses detected in antigen-stimulated peripheral blood mononuclear cells did not differ between the three groups when the comparison was performed without considering HLA genotypes. However, significant differences were observed, when children with the high-risk genotype HLA (DRB1*03)-DQA1*05-DQB1*02/DRB1*0401-DQA1*03-DQB1*0302 were analyzed separately. The responses to the peptides including amino acids A1-12 derived from B1 and H1 were significantly higher in children with T1D (P=0.008, P=0.004, for B1 and H1, respectively) and in children with diabetes-associated autoantibodies (P=0.002 and P=0.001, respectively) than in control children. Positive responses (stimulation indices SI> or =3) were seen more frequently in T1D children than in controls (4/7 vs 2/19; P=0.03 and 4/7 vs 1/19; P=0.01 for B1 and H1, respectively). T-cell response to the insulin peptide A1-12 is enhanced in clinical and preclinical T1D associated with the high-risk HLA-genotype emphasizing the importance of this epitope.