Enterovirus infections, implicated in the pathogenesis of type 1 diabetes in a number of studies, may precipitate the symptoms of clinical diabetes and play a role in the initiation of the beta-cell damaging process. The aim of this study was to evaluate whether cellular immune responses to enterovirus antigens are abnormal in children with type 1 diabetes. Lymphocyte proliferation responses to enterovirus antigens were analyzed in 41 children with new-onset type 1 diabetes, 23 children with type 1 diabetes for 4-72 months, and healthy control children in subgroups matched for HLA-DQB1 risk alleles, sex, and age. Children with diabetes for 4-72 months more often had T-cell responses to the Coxsackievirus B4-infected cell lysate antigen than children with new-onset diabetes (P < 0.01) or control children (P < 0.01). Responses to recombinant nonstructural protein 2C of Coxsackievirus B4 were also more frequent in children with type 1 diabetes for 4-72 months when compared with control subjects (P = 0.03), whereas the responses to purified Coxsackievirus B4 and recombinant VP0 protein, which did not contain nonstructural proteins, did not differ. These data suggest that T-cell responses to Coxsackievirus B4 proteins and particularly to the antigens containing the nonstructural proteins of the virus are increased in children with type 1 diabetes after the onset of the disease. However, in children with new-onset diabetes, responses were normal or even decreased. This phenomenon was specific for enteroviruses and could be caused by trapping of enterovirus-specific T-cells in the pancreas.