Pyruvate kinase activity in primary cultures of hepatocytes isolated from a normal rat was maintained at a constant level similar to that found in vivo (14.0 +/- 2.8 units per mg of DNA) for over 6 days when both dexamethasone and insulin were included in the medium. Yet the pyruvate kinase activity decreased 50% when the cells were cultured for 2 days and 4 days, respectively, in the presence of either dexamethasone or insulin alone. A brief, 10 min incubation of hepatocytes in the presence of dexamethasone was sufficient to maintain the enzyme activity of cells subsequently cultured for 4 days in the presence of insulin. The optimal dexamethasone concentration was 1 microM. Three other glucocorticoids were able to maintain the pyruvate kinase activity in cells cultured in medium containing insulin. The presence of the protein synthesis inhibitors, actinomycin D or cyclohexamide in cells cultured in the presence of dexamethasone and insulin resulted in a 25% decrease in the pyruvate kinase activity. Therefore, it is suggested that the synergistic effect of glucocorticoids and insulin to maintain pyruvate kinase activity in primary cultures of hepatocytes is dependent upon the ability of these cells to maintain protein synthesis.