The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-13C]glucose and [2-13C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the 13C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-13C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.