Organ cultures prepared from 18- to 19-day fetal and 3- to 6-day-old newborn rat liver were maintained for 2 days in Trowell's T8 medium without insulin and supplemented with 0.1% albumin and 300 mg% glucose. The atmosphere for culture was 95% O2 and 5% CO2. Medium alone was used for control cultures, whereas insulin, hydrocortisone, or insulin plus hydrocortisone were used in experimental groups. Explant glycogen stores were maintained better in cultures grown in hormone-supplemented media than in control cultures. Fetal explants were found to have higher levels of glycogen than controls in the insulin or insulin plus hydrocortisone groups. Postnatal explants did not have higher levels of glycogen in the hormone-treated groups. Gestational age appeared to determine whether the liver explants reacted to hydrocortisone or insulin to maintain glycogen stores. Enzymatic assays, in vitro of glycogen synthetase and phosphorylase, indicated that the fetal liver response to insulin plus hydrocortisone by increasing the total and independent form of glycogen synthetase; but similar enzyme studies on postnatal rat liver did not show convincing differences as to an effect on synthetase. No definite in vitro temporal relationships could be identified. Late in gestation, the effect of hydrocortisone on glycogen synthesis is apparently dependent on the presence of insulin. Insulin appears to be required for glycogen storage in vitro in the cultures of postnatal rat liver.