Analysis of the two-dimensional electrophoretic patterns of total radiolabeled cellular proteins derived from human orbital fibroblast cultures revealed that interferon gamma (100 U/ml) elicited significant quantitative changes in 42% of 86 randomly-selected proteins relative to untreated cultures. The most substantial up-regulation involved a protein with pI/mw map coordinates of 5.9/54,000 and a heterogenous 5 isoform protein cluster (pIs = 6.1-5.6) of approximately 47- to 50-kDa. These proteins were identified as the previously described 54-kDa protein inducible in interferon gamma-sensitive cell types and type-1 plasminogen activator inhibitor (PAI-1), respectively. Definition of PAI-1 as an interferon gamma-responsive protein in orbital fibroblasts was confirmed by immunoprecipitation using PAI-1-specific antibodies. Induction of PAI-1 and the 54-kDa protein in orbital fibroblasts, moreover, was relatively specific for interferon gamma since interferon alpha failed to initiate a similar inductive response. The synthesis of a 170 kDa protein, tentatively identified as a collagen, was decreased by approximately 80%. Analysis of the labeled proteins secreted into the culture medium revealed that interferon gamma increased the medium content of fibronectin and decreased the secretion of collagen. It would appear from these data that the inflammatory cytokine can exert regulatory effects on the synthesis of many specific proteins in orbital fibroblasts.