Gluconeogenesis from 10 mM lactate has been studied in isolated hepatocytes from fetal, newborn, and 70-day-old rabbits. Gluconeogenesis proceeds to a very low rate in fetal rabbit hepatocytes despite substantial activities of all gluconeogenic enzymes including mitochondrial phosphoenolpyruvate carboxykinase. A tenfold increase in the rate of gluconeogenesis occurs in hepatocytes from 1- or 2-day-old fasting or suckling newborn rabbits. The emergence of gluconeogenic capacity in newborn rabbit hepatocytes is triggered by birth itself and not by a chronological factor, and it is primarily controlled by an increase in the activity of cytosolic phosphoenolpyruvate carboxykinase. Moreover, an active fatty acid oxidation is essential to support a high rate of gluconeogenesis in hepatocytes from newborn rabbits.