The present study determined the metabolic fate of [U-13C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain and also in cultures incubated in the presence of 1 or 5 mM α-ketoisocaproate (α-KIC). When astrocytes were incubated with 0.2 mM [U-13C]glutamate, 64.1% of the 13 C metabolized was converted to glutamine, and the remainder was metabolized via the tricarboxylic acid (TCA) cycle. The formation of [1,2,3-13C3]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. In control astrocytes, 8.0% of the [13C]glutamate metabolized was incorporated into intracellular aspartate, and 17.2% was incorporated into lactate that was released into the medium. In contrast, there was no detectable incorporation of [13C]glutamate into aspartate in astrocytes incubated in the presence of α-KIC. In addition, the intracellular aspartate concentration was decreased 50% in these cells. However, there was increased incorporation of [13C]glutamate into the 1,2,3-13C3-isotopomer of lactate in cells incubated in the presence of α-KIC versus controls, with formation of lactate accounting for 34.8% of the glutamate metabolized in astrocytes incubated in the presence of α-KIC. Altogether more of the [13C]glutamate was metabolized via the TCA cycle, and less was converted to glutamine in astrocytes incubated in the presence of α-KIC than in control cells. Overall, the results demonstrate that the presence of α-KIC profoundly influences the metabolic disposition of glutamate by astrocytes and leads to altered concentrations of other metabolites, including aspartate, lactate, and leucine. The decrease in formation of aspartate from glutamate and in total concentration of aspartate may impair the activity of the malate-aspartate shuttle and the ability of astrocytes to transfer reducing equivalents into the mitochondria and thus compromise overall energy metabolism in astrocytes.