Elevated glutamate levels have been reported in humans with diabetic retinopathy. Retinal Müller glial cells regulate glutamate levels via the GLAST transporter and system x_c ⁻ (cystine-glutamate exchanger). We have investigated whether transporter function and gene and/or protein expression are altered in mouse Müller cells cultured under conditions of hyperglycemia or oxidative stress (two factors implicated in diabetic retinopathy). Cells were subjected to hyperglycemic conditions (35 mM glucose) over an 8-day period or to oxidative stress conditions (induced by exposure to various concentrations of xanthine:xanthine oxidase) for 6 h. The Na⁺-dependent and –independent uptake of [³H] glutamate was assessed as a measure of GLAST and system x_c ⁻ function, respectively. Hyperglycemia did not alter the uptake of [³H] glutamate by GLAST or system x_c ⁻; neither gene nor protein expression decreased. Oxidative stress (70:14 or 100:20 μM xanthine:mU/ml xanthine oxidase) decreased GLAST activity by ~10% but increased system x_c ⁻ activity by 43% and 89%, respectively. Kinetic analysis showed an oxidative-stress-induced change in V_max, but not K_m. Oxidative stress caused a 2.4-fold increase in mRNA encoding xCT, the unique component of system x_c ⁻. Of the two isoforms of xCT (40 and 50 kDa), oxidative stress induced a 3.6-fold increase in the 40-kDa form localized to the plasma membrane. This is the first report of the differential expression and localization of xCT isoforms as caused by cellular stress. Increased system x_c ⁻ activity in Müller cells subjected to conditions associated with diabetic retinopathy may be beneficial, as this exchanger is important for the synthesis of the antioxidant glutathione.