Orbital fibroblasts in culture display phenotypic attributes that distinguish them from fibroblasts derived from other anatomical regions. The current studies were conducted to define potential cellular heterogeneity among orbital fibroblasts with regard to 1) differential expression of Thy-1, a 25-kilodalton glycoprotein associated with cell signaling; 2) cells undergoing a change in shape in response to prostaglandin E2 (PGE2); and 3) differences in morphology and Thy-1 expression between single cell-derived clonal fibroblast strains. On the basis of flow cytometric analysis using an anti-Thy-1 monoclonal antibody, 65% of intact orbital fibroblasts expressed surface Thy-1 (n = 5; range, 54-71%). In contrast, greater than 95% of the fibroblasts present in the five dermal strains tested were Thy-1 positive. A total of six strains of orbital fibroblasts were assessed for their shape change response to a 4-h treatment with PGE2 (100 nmol/L). A mean of 37% of the fibroblasts present in each culture responded to PGE2 (range, 22-50%). In contrast, only 1% of dermal fibroblasts exhibited any change in morphology. Three separate clones were generated from a single parent strain of Graves' orbital fibroblasts. These clones consisted of homogeneous appearing cells; however, substantial clone to clone differences in morphology were stably expressed for several population doublings. Thy-1 was expressed uniformly in cells of two clones, whereas the third was Thy-1 negative. Factor VIII and smooth muscle-specific alpha-actin were undetectable in any of the orbital or dermal cultures examined. Thus, Thy-1 expression is uniform in fibroblasts from certain anatomical regions such as the skin and heterogeneous in cells derived from human lung and orbit. These findings suggest that human orbital connective tissue may have a complexity not previously appreciated.