Purpose: To explore the immune mechanism of Graves ophthalmopathy (GO) by analyzing infiltrating cells in orbital connective tissue (OCT) specimens of patients with active GO using immunohistochemical methods. Methods: Five OCT specimens obtained from patients with active GO and five control specimens obtained from forensic cadavers who died from nonmedical reasons were stained with anti-CD3, CD4, CD8, CD45RO, HLA-Dr, CD25, and TNF-alpha monoclonal antibodies. Positively stained cells were counted and results were interpreted as cell counts/mm(2). Four of five GO patients had never been treated with any immunomodulating therapy. Only one had received oral prednisolone prior to tissue sampling, but this treatment had ceased 5 months before surgery. Results: The retro-orbital tissue specimens obtained from forensic cadavers did not show any significant positive staining for any monoclonal antibody tested. However, the specimens from GO patients showed positively stained means of 36.66 +/- 4.61 HLA- Dr(+), 12.8 +/- 3.42 CD8(+), 11.8 +/- 1.78 CD4(+), 16.6 +/- 1.81 CD3(+), 21.2 +/- 3.12 CD45RO(+), 10.4 +/- 2.07 TNF-alpha(+), 7.2 +/- 1.48 CD25(+), 3.2 +/- 1.09 CD4(+) CD8(+), 4.6 +/- 1.67 CD4(+) CD45RO(+), 2.8 +/- 0.83 CD8(+) CD45RO(+), 1.6 +/- 0.89 CD4(+) CD25(+), and 1.8 +/- 1 0.83 CD8(+) CD25(+) cells/ mm(2). Conclusions: Our study supports that most of the infiltrating lymphocytic cells in the active stage of GO are T cells, and a significant proportion of them are CD45RO+ cells. Infiltration of OCT by HLA- Dr+, CD25+, and TNF-alpha cells suggests that Th1-type immune reaction with the interference of proinflammatory cytokine(s) (TNF-alpha) may be important in the pathogenesis of disease. Further studies are needed to understand the disease pathogenesis and may provide a scientific basis for future treatment alternatives for the disease (e. g., anticytokine treatment).