Organ cultures prepared from 3- to 6-day-old newborn rat liver were maintained for as long as 6 days in medium CMRL-1066 supplemented with horse serum, glucose, and L-glutamine; the atmosphere for culture was 95 per cent O2 and 5 per cent CO2. Medium alone was used for control cultures, whereas insulin, hydrocortisone, or insulin plus hydrocortisone were used in experimental groups. Explant glycogen stores and viability were better preserved in cultures grown in hormone-supplemented media than in control cultures. Insulin caused glycogen storage of explant glycogen. Results of light and electron microscopic abservations are confirmed by isotope studies. The effect of hydrocortisone on glycogen synthesis is apparently dependent on the presence of insulin, and insulin appears to be required for glycogen storage in vitro in these cultures of postnatal rat liver.