The molecular basis for the profound inflammatory response and the accumulation of hyaluronan in orbital connective tissues seen in thyroid-associated ophthalmopathy is unknown. Moreover, the link between the orbital manifestations of Graves’ disease and those in the pretibial skin, localized dermopathy, has yet to be established. We have reported recently that leukoregulin, an activated T lymphocyte-derived cytokine, dramatically induces hyaluronan synthesis and prostaglandin–endoperoxide H synthase 2 in human orbital fibroblasts in culture. In the current studies, utilizing giant two-dimensional gel electrophoresis, we find that orbital fibroblasts express constitutively a protein profile that distinguishes them from skin fibroblasts derived from the abdominal wall and from the pretibium. We further demonstrate that leukoregulin, when present in culture medium for 16 hr, up-regulates a set of orbital fibroblast proteins not present in untreated cultures or in fibroblasts from the abdominal wall. However, some of the same protein inductions are present in the pretibial fibroblasts. These leukoregulin-induced changes in protein expression are completely blocked by dexamethasone (10 nM). Our findings are the first to identify proteins that appear to be expressed and differentially regulated in an anatomical site-restricted manner in orbital and pretibial fibroblasts and seem to establish a molecular link between fibroblasts from the orbit and those in pretibial skin.