Histologic examination of the retroocular connective tissues in Graves' ophthalmopathy (GO) reveals lymphocytic infiltration and an accumulation of glycosaminoglycans (GAG), hydrophilic macromolecules produced locally by fibroblasts. We studied the in vitro effect on fibroblast GAG production of several cytokines and growth factors likely to be secreted by these activated lymphocytes or macrophages. Cultures were established from retroocular connective tissue, extraocular muscle perimysium, and pretibial skin obtained from patients undergoing orbital decompression or eye muscle surgery for severe GO and from normal individuals. Confluent cultures were treated with one of the compounds and labeled with [3H]glucosamine or [35S]sulfuric acid for quantitation of [3H]GAG or [35S]GAG accumulation. Of the various compounds examined, only interleukin-1 (IL-1) and transforming growth factor (TGF)-beta significantly stimulated [3H]GAG accumulation in a dose- and time-dependent fashion. There was no difference in sensitivity to the GAG-stimulating effect of IL-1 or TGF-beta between fibroblasts from the four anatomical sites studied or between normal and GO patient fibroblasts. In conclusion, both IL-1 and TGF-beta are potent stimulators of [3H]GAG accumulation by retroocular connective tissue and perimysial fibroblasts, as well as by fibroblasts from the dermal sites studied. Stimulation of GAG production by these cytokines, released from lymphocytes or macrophages infiltrating the retroocular space, may play a role in the accumulation of GAG in the retroocular and perimysial connective tissues in GO.