The system x(C)- (Sx(C)-) transporter functions to mediate the exchange of extracellular cystine (L-Cys(2)) and intracellular glutamate (L-Glu). Internalized L-Cys(2) serves as a rate-limiting precursor for the biosynthesis of glutathione (GSH), while the externalized L-Glu can contribute to either excitatory signaling or excitotoxicity. In the present study the influence of culture conditions (with and without dibutyryl-cAMP) and GSH levels on the expression of Sx(C)- were investigated in primary rat astrocyte cultures. Sx(C)- activity in dbcAMP-treated cells was nearly sevenfold greater than in untreated astrocytes and increased further (∼threefold) following the depletion of intracellular GSH with buthionine sulfoximine. This increase in Sx(C)- triggered by GSH depletion was only observed in the dbcAMP-treated phenotype and was distinct from the Nrf2-mediated response initiated by exposure to electrophiles. Changes in Sx(C)- activity correlated with increases in both protein and mRNA levels of the xCT subunit of the Sx(C)- heterodimer, an increase in the V(max) for L-Glu uptake and was linked temporally to GSH levels. This induction of Sx(C)- was not mimicked by hydrogen peroxide nor attenuated by nonspecific antioxidants but was partially prevented by the co-administration of the cell-permeant thiols GSH-ethyl ester and N-acetylcysteine. These findings demonstrate that the expression of Sx(C)- on astrocytes is dynamically regulated by intracellular GSH levels in a cell- and phenotype-dependent manner. The presence of this pathway likely reflects the inherent vulnerability of the CNS to oxidative damage and raises interesting questions as to the functional consequences of changes in Sx(C)- activity in CNS injury and disease.