Graves' ophthalmopathy (GO), in which orbital tissues are infiltrated with activated T lymphocytes and hyaluronan, can manifest an overabundance of adipose tissue in the human orbit. Little is known about adipogenesis in this anatomic region. We have investigated whether orbital fibroblasts in culture possess the capacity to undergo adipocytic differentiation. Orbital tissue from patients with or without GO was placed in primary culture, and proliferating fibroblasts were sub-passaged. Confluent fibroblasts were subjected to a differentiation protocol, involving a serum-free defined medium supplemented with insulin, triiodothyronine, carbaprostacyclin, thyrotropin, dexamethasone, and isobutylmethylxanthine. Control cells were maintained in serum-free medium supplemented only with insulin. After approximately 14 days, light microscopy revealed characteristic morphologic changes of adipocyte differentiation, including cell rounding and lipid droplet accumulation. Oil Red O staining could be demonstrated in those cells. Scanning electron microscopy revealed that the cells undergoing adipogenesis contain multiple, discrete lipid droplets. The overall percentage of fibroblasts undergoing differentiation was somewhat variable, but no more than 5-10%. The adipocytes arise in close proximity to each other, and often at the periphery of the culture surface. In contrast, dermal fibroblasts and perimysial fibroblasts from extraocular muscle fail to differentiate. We conclude that a subpopulation of orbital fibroblasts is capable of adipocyte differentiation. These in vitro observations may represent the in vivo process that contributes to excess orbital adipose tissue volume in GO.