Insulin is widely used as a growth factor in hepatocyte culture but its effect on the production of acute-phase proteins has not been studied. By measuring four positive (fibrinogen, alpha 1-antitrypsin, alpha 1-acid glycoprotein, and alpha 1-antichymotrypsin) and four negative (albumin, prealbumin, transferrin, and retinol binding protein) acute-phase proteins produced by the Hep G2 hepatoma cell line, we have shown that insulin is an important modulator of acute-phase protein production. Our data show that insulin is able to inhibit the synthesis of prealbumin, transferrin, and fibrinogen. The results also show a complex interaction between insulin, interleukin 6, and glucocorticoids because insulin is able to inhibit the dexamethasone induction of alpha 1-antichymotrypsin, and in the presence of interleukin 6, dexamethasone is able to regulate the production of fibrinogen and prealbumin. The regulatory role of insulin in fibrinogen production was confirmed by pulse chase labeling followed by immunoprecipitation and fluorography.