Optimum conditions were established for the assay of glycogen, glycogen synthase, glycogen phosphorylase, phosphoglucomutase, and glucose-6-phosphatase in rabbit fetal heart, lung, and liver. Using these methods, the pattern of appearance of glycogen and the above four enzymes was established from day 18 of gestation to day 8 after birth. The results indicate that total tissue glycogen reaches maximum levels between days 22 and 24 in the heart, days 24 and 26 in the lung, and days 30 and 31 in the liver. In all three tissues, the rapid rise or depletion of glycogen is coincident with a corresponding increase in glycogen synthase and glycogen phosphorylase activities. However, substantial amounts of glycogen synthase are present both prior to and after the accumulation of glycogen. Similarly, considerable amounts of glycogen phosphorylase are present early in gestation, yet deposition of glycogen occurs. Both the I and D forms of glycogen synthase are present in the three tissues, the major being the physiologically inactive D form. Similarly both the a and b forms of glycogen phosphorylase are present, with the a form (active form) making up about 30-60% of the total phosphorylase activity. Glucose-6-phosphatase was absent in fetal heart and lung throughout the period of gestation investigated. Low levels of this enzyme were detectable in fetal liver near term. The phosphoglucomutase activity increased progressively from day 22 of gestation in all three tissues and continues to increase after birth. The disappearance of fetal lung glycogen occurs between days 27 and 28 at a time when surfactant phospholipids first appear. These findings indicate that the breakdown of glycogen is providing the fetal lung cells with energy necessary for surfactant phospholipid biosynthesis.