Orbital fibroblasts from patients with Graves' ophthalmopathy, when treated with prostaglandin E2 (PGE2), become stellate and develop prominent cellular processes. In this paper, we describe results of studies designed to characterize the action of PGE2 on orbital fibroblast shape changes in vitro. Orbital and dermal fibroblasts were incubated with PGE2, one of several prostanoid analogues, 8-br-cAMP or forskolin and were then visualized by phase-contrast microscopy. Other studies involved seeding cells in special chambers equipped with electrodes for cell sensing using electric cell-substrate impedance sensing (ECIS) to detect changes in shape. PGE2 (10(-7) mol/L) elicited a rapid and dramatic alteration in the shape of orbital fibroblasts but not those derived from the skin. Cells became stellate and developed prominent cytoplasmic processes that extended out from the central area containing the cell nucleus. The effects were stereoselective in that a number of structurally related compounds, including Sulprostone, PGI2, PGF2 alpha, thromboxane A2, thromboxane B2, and 11 deoxy,16,16 dimethyl PGE2 failed to elicit a similar shape change. Butaprost (10(-5) mol/L), a specific EP2 agonist, elicited a similar shape-change as that observed with PGE2. 16,16-dimethyl PGE2, a nonselective agonist, could mimic the action of PGE2. The effect of PGE2 was apparent at 10(-8) mol/L, maximal at a concentration of 10(-7) mol/L and took 4-8 hr to evolve completely. Cycloheximide (10 micrograms/mL) and actinomycin D (1 micrograms/mL) failed to block the shape change. The morphologic change could be reproduced by addition of 8-br-cAMP (3 mmol/L) and by forskolin (5 mumol/L). Moreover, PGE2 and Butaprost treatment elicited in orbital cultures a massive increase in endogenous cAMP production while analogues not affecting cell shape failed to influence cyclic nucleotide generation. Three strains of orbital fibroblasts from patients with Graves' ophthalmopathy and three from normal orbits were tested and all responded to PGE2 (10(-7) mol/L). Four strains of dermal fibroblasts failed to respond to PGE2. The changes in orbital fibroblast morphology were accompanied by a marked decrease in monolayer impedance as assessed by electric cell-substrate impedance sensing. The earliest effects were apparent within 30 min using this sensitive technique. The widely recognized roles of PGE2 and related compounds in the mediation of the inflammatory response make our current findings in orbital fibroblasts of potential importance to the pathogenesis of Graves' ophthalmopathy.