The effect of the glucocorticoids, insulin, and glucose concentration on glycogen deposition in adult rat liver parenchymal cells maintained in a chemically defined, serum-free medium has been studied. Increasing the medium concentration of glucose from 5.6 mM to 30.6 mM in the absence of hormones increased cellular glycogen content from 6.5 to 51 micrograms of glycogen per mg of cell protein. Treatment of the cells with insulin increased the glycogen content by 15 to 30% at medium glucose concentrations above 10.6 mM. The addition if the synthetic glucocorticoid, dexamethasone, to the culture medium resulted in 40 to 105% increased in glycogen content at glucose concentrations greater than 5.6 mM. The addition of dexamethasone and insulin together in the culture medium resulted in an increase in glycogen content that was greater than the additive effect of each hormone alone. This study establishes that glucose concentrations above 10.6 mM stimulate glycogen deposition in the absence of any hormonal stimulus. In addition, glucocorticoids directly stimulate glycogen deposition at glucose concentrations which are greater than physiological (5.6 mM).