The rate of glutaminase-dependent metabolism of glutamine in intact astrocytes was determined under conditions in which the extracellular concentration of glutamine was varied between 0.2 and 3.2 mM glutamine for control and dibutyryl cyclic AMP (dBcAMP)-treated cells. Glutamine metabolism by intact cells increased with increasing extracellular glutamine when calculations were based on the extracellular specific activity of glutamine. However, when the rate was based on the intracellular specific activity of glutamine, the rate of glutamine metabolism was independent of the media glutamine concentration. Similar results were obtained when cells were treated with dBcAMP, although the rates were approximately twice as high compared to untreated cells. The rate of formation of 14CO2 from [1-14C]glutamine and [1-14C]glutamate, based on the extracellular specific activities, were 93 +/- 5 and 40 +/- 4 nmol/mg protein/h, respectively. Oxidation rates based on the experimentally determined intracellular specific activity of glutamine and glutamate were 144 +/- 8 and 209 +/- 18 nmol/mg protein/h, respectively. In dBcAMP-treated astrocytes, the oxidation rates were higher than in untreated cells. These studies demonstrate that determination of the specific activity of compounds inside the cell aids in the interpretation of metabolic studies with intact cells and that both the initial steps of glutamine metabolism and the rate of 14CO2 formation from 14C-glutamine via the TCA cycle were increased in dBcAMP-treated astrocytes.