The glucose concentration in the culture medium may affect the energy metabolism of cultured cells. The oxidative metabolism of glucose in astrocytes might also be affected because the glucose concentration (25 mmol/L) of many culture media is higher than the physiological levels (∼3 mmol/L). In the present study, we assessed the effects of reducing the glucose concentration in the culture medium on the oxidative metabolism of glucose in cultured rat astroglia by measuring the oxidation rates of L-[U-¹⁴C]lactate or D-[U-¹⁴C]glucose to ¹⁴CO₂. The effects of D-aspartate and elevated extracellular K⁺ levels on oxidative and glycolytic metabolism in astroglia were also investigated. The rates of [¹⁴C]lactate and [¹⁴C]glucose oxidation in astroglia cultured in a medium containing 2 mmol/L of glucose (astroglia₂) were approximately twofold of those in astroglia cultured in a medium containing 22 mmol/L of glucose (astroglia₂₂). D-Aspartate (500 μmol/L) significantly increased [¹⁴C]lactate oxidation by 156% in astroglia₂₂ and by 83% in astroglia₂. D-[U-¹⁴C]glucose oxidation in astroglia₂₂ and astroglia₂ was also increased by 94% and 76%, respectively. In contrast, an elevated extracellular K⁺ concentration (7.4 mmol/L) did not affect glucose and lactate oxidation, although it increased 2-deoxy-D-[1-¹⁴C]glucose phosphorylation. Astroglia grown in the physiological glucose concentration are more dependent on the oxidative metabolism of glucose than that in high-glucose concentration. Glucose concentration in culture medium has a strong influence on astrocytic oxidative capacity in vitro.