The cytotoxic T lymphocyte antigen-4 (CTLA-4) (CD152) is a basic negative regulatory molecule of T cell activation and its hypo-function is associated with severe lymphoproliferative syndrome. The aim of the present study was to evaluate the intracellular and surface expression of CTLA-4 on peripheral T cells before and after T cell activation in children with Hashimoto's thyroiditis (HT). Blood samples were obtained from 46 children: 25 with Hashimoto's thyroiditis and 21 controls free of autoimmune disease or thyroid disorders. T cell phenotype was evaluated by flow cytometry with the use of monoclonal antibodies combination: CD4- FITC/ CD28 -PC5/ CD152 -PE and CD8 -FITC/ CD28 -PC5/ CD152 -PE on T cell surface and intracellularly at baseline and after 48 h of T cell culture with the mitogen 48-PHA. We found that the number of T cells with intracellular CD152 expression was comparable in HT patients and controls at baseline and increased after 48-PHA, in CD4 subset only, in both patients and controls. However, the increase was more evident in the HT patients. The number of T cells with the surface expression of CD152 at baseline was significantly lower in the HT patients than in controls (p < 0.0002) in non-stimulated CD4+ and CD8+ T cells. After 48-PHA, surface CD152 expression in CD4+T cells increased in both groups; the increase was greater in controls. In conclusion, impaired function of CTLA-4 in HT patients may depend on the imbalance of intracellular/surface expression of CD152 in T cells.