BACKGROUND AND AIM:: Gravesʼ orbitopathy (GO) is characterized by orbital T cell infiltration and local release of proinflammatory cytokines. We aimed to evaluate the involvement of baseline regulatory T (Treg) cells and rabbit anti-T lymphocyte globulin (rATG)-induced Treg cells in GO. DESIGN:: Peripheral blood mononuclear cells (PBMCs) from seven patients with Gravesʼ disease (GD) without eye manifestations, 29 patients with GO, and 15 healthy controls were incubated with rATG, washed, and analyzed for expression of Treg cell markers and for ability to suppress mixed lymphocyte reaction. RESULTS:: Elevation of CD4 to CD8 ratio and enhanced secretion of IL-6, IL-10, and TNFα were detected in PBMCs of GO patients compared with controls (both P < 0.01). Despite this abnormality, the frequencies of CD4CD25FoxP3 of GO and control PBMCs were similar and remained unchanged after 24 h incubation with control rabbit IgG (rIgG). Incubation with polyclonal rATG increased the frequency of PBMCs of GO patients, expressing Treg cell markers (CD25, FoxP3, and the IL-7 receptor CD127) by 2.5–8 fold over corresponding rIgG-incubated cells (P < 0.05). FoxP3/CD4 rATG-induced Treg cell marker expressed more intensively on GO peripheral blood leukocytes (PBLs) than on GD (P < 0.01) or normal (P < 0.05) PBLs, yet its expression on normal PBLs was stronger than on GD PBLs (P < 0.05). GO rATG-incubated PBMCs, but not rIgG-incubated PBMCs, suppressed (P < 0.05) proliferation of autologous responder cells stimulated with allogeneic irradiated cells in mixed lymphocyte reaction. Such rATG-induced suppressive activity was not detected in GD. CONCLUSION:: This study is the first to show that PBMCs of patients with GO substantially increase Treg cells in both frequency and potency after in vitro incubation with rATG.