We hypothesize that fibroblasts obtained from the retroocular space and the pretibial skin, sites affected by the peripheral manifestations of Graves' disease, share unique characteristics that may in part explain the site specificity of Graves' ophthalmopathy (GO) and pretibial myxedema (PTM). Heat shock proteins (HSPs), synthesized by cells undergoing stress, function to maintain cellular homeostasis and are probably involved in the intracellular processing and cell surface presentation of antigens. We investigated possible differences in the expression of 70-kDa HSPs between cultured fibroblasts obtained from patients with severe GO and normal individuals. In addition, we compared HSP expression in fibroblasts derived from tissues involved in the extrathyroidal manifestation of Graves' disease (GO and PTM) with that in fibroblasts from uninvolved tissues. HSPs were detected by both immunoblotting and indirect immunofluorescence, using monoclonal antibodies that are directed against HSP72, HSP72/73 (termed HSP70), and HSP90. HSP expression at baseline and after treatment with various cytokines and heat stress was examined. At baseline, HSP72 reactivity was exclusively detected in retroocular and pretibial fibroblasts from patients with severe GO and PTM, but was not observed in abdominal fibroblasts from these patients and was not detectable in fibroblasts from any anatomical site of normal individuals. The abundance of HSP70 expression at baseline and after treatment with certain cytokines was significantly greater in retroocular and pretibial fibroblasts from patients with GO than in normal individuals. In addition, characteristic changes in the cellular localization of HSPs before and after exposure to heat stress and cytokines were observed; cell surface expression of HSP70 was detected at baseline in fibroblasts from patients, but not in normal fibroblasts. These data provide the first evidence that HSPs are differentially expressed by fibroblasts derived from tissues affected by the extrathyroidal manifestations of GD. These proteins may have a role in localized immune processes, leading to the development of GO and PTM.