We have established previously that the adult rat hepatocyte produces collagen under conditions of primary monolayer culture or under pathologic conditions in vivo. During the initial 3 days of incubation of hepatocytes in serum-free culture medium, collagen synthesis represented only 0.01%-0.05% of the synthesis of noncollagen proteins. Thereafter, rates of collagen synthesis rose dramatically, and after 9 days of incubation were increased an average tenfold in cultures derived from normal hepatocytes and 30-fold in cultures derived from regenerated liver. The spontaneous increase in the rates of collagen synthesis was almost completely blocked (94%) when cultures were incubated for 8 days in medium containing the synthetic glucocorticoid dexamethasone (10(-7) M). Dexamethasone was without effect on the synthesis of noncollagen proteins. Inhibition of collagen synthesis by dexamethasone exhibited a sigmoidal dose-response curve with 50% inhibition observed at a concentration of 10(-9) M. Collagen synthesis was diminished similarly by other glucocorticoids (hydrocortisone, methylprednisolone, triamcinolone, prednisone, all 10(-6) M) but not by other hormonal or nonhormonal steroids (progesterone, estradiol, testosterone, all 10(-5) M). We excluded increased collagen degradation as an explanation for these results by demonstrating that dexamethasone failed to increase the incorporation of radioactive proline into trichloroacetic acid-soluble radioactive hydroxyproline. Exposure of hepatocytes to dexamethasone during the first 48 h of incubation produced inhibition of collagen synthesis in 9-day-old cultures, even though washout studies with [3H]dexamethasone revealed negligible retention of the steroid by the cells. This observation suggests that among the initial, culture-adaptive responses of the hepatocyte are glucocorticoid-sensitive events that culminate in increased rates of collagen synthesis days later. We conclude that glucocorticoids may serve as natural suppressors of the potential of the hepatocyte to produce collagen.