Astroglia-rich primary cultures derived from the brains of newborn rats can be grown in the presence of sorbitol or fructose. In the present study, evidence was obtained by enzymatic analysis and immunocytochemistry that fructose is further metabolized to fructose-6-phosphate and that fructokinase is lacking in the astrocytes. In contrast, fructose-1-phosphate as well as fructokinase immunoreactivity could be detected in cultured hepatocytes. Considerable amounts of astroglial glycogen were synthesized from fructose. Lactate release in fructose-fed cultures was still 30% that of glucose-fed cells and was abolished in the presence of 2-deoxyglucose. No glycogen was synthesized when sorbitol, which is converted intracellularly to fructose, replaced glucose in the incubation medium. However, lactate release from sorbitol-fed cultures was still significant and was not abolished by 2-deoxyglucose. The results are compatible with the idea of astroglial glycogen being a store of lactate rather than glucose. Furthermore, the results demonstrate that sorbitol is an adequate substrate for astroglial glycolysis but, in contrast to fructose, cannot be utilized for the buildup of glycogen stores.