In Graves' ophthalmopathy (GO), retrobulbar connective tissue is infiltrated by T cells whose role in the pathogenesis of the disease was investigated in the present work. The aims included firstly to characterize subsets of blood lymphocytes and of sessile lymphocytes cloned from a retroorbital tissue specimen. Second, in counterstimulation assays, the ability of patients' T cells to influence cultivated retrobulbar fibroblasts and in turn the enhancement of lymphocyte proliferation by retrobulbar fibroblasts was investigated. Blood lymphocytes of 16 GO patients and 12 controls isolated by density gradient centrifugation and retrobulbar fibroblasts obtained from orbital decompression were alternately exposed to irradiation of 4000 rad (to suppress proliferation of either cell type), then cocultivated for 48 h. Subsequently, the cells (250,000 lymphocytes, 5000 retrobulbar fibroblasts) were incubated with [3H]thymidine for 24 h. A stimulation index representing the degree of proliferation in comparison with a control was determined. Screening of 62 retrobulbar lymphocyte clones by cell-ELISA revealed a CD4/CD8 ratio of 8.2, contrasting with a normal ratio of 2.1 in peripheral lymphocytes (as obtained by FACS analysis). Incubation of patient's lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly elevated stimulation index (9.7) compared to incubation of lymphocytes with retrobulbar fibroblasts from controls (2.5). In another assay, the degree of stimulation amounted to 2.2 vs. -0.8. Retrobulbar fibroblasts were stimulated twice as much by lymphocytes of the same patient compared to retrobulbar fibroblasts from controls. The autologous reactions demonstrated suggest the presence of autoreactive T lymphocytes in GO patients directed against retrobulbar fibroblasts, which, in addition, are stimulated by these lymphocytes.