An increase of retrobulbar adipose tissue has been shown by imaging techniques in patients with Graves' ophthalmopathy (GO). Immunohistochemical staining was applied to investigate the involvement of different retrobulbar (especially adipose) tissue components in the autoimmune process of the disease. Cryostat sections from retrobulbar tissues of 15 GO patients and 11 controls were analyzed with a battery of monoclonal antibodies against CD2, CD4, CD8, CD11a, CD19/22, CD25, CD54, CD57, CD68, C3b, HLA-A, B, C, and HLA-DR. In contrast to controls, the retrobulbar adipose tissue showed an increase of HLA-DR expression, an activation of intercellular adhesion molecule I (ICAM-1, CD54), as well as a marked infiltration of activated T-cells (especially CD4) and macrophages (CD68), whereas a positive staining for interleukin-2 receptors (CD25), T-cells (CD2), B-cells (CD19/22), complement factors (C3b), and LFA-1 (CD11a) was present only in a few patients' specimens. Staining for natural killer cells (CD57) was negative in fat tissue of patients and controls. The perimuscular connective tissue sections of GO patients showed an accumulation of T-cells (CD4/CD8), an enhanced display of ICAM-1, LFA-1, HLA-DR-positive cells, and B-cells. Retrobulbar muscle tissue of GO patients exhibited an increase of CD4, CD8, CD54, CD68, and HLA-DR-positive cells, located in the intramuscular connective tissue, exclusively. In conclusion, the present study underlines the involvement of retrobulbar adipose tissue in the immunopathogenesis of Graves' ophthalmopathy.