AbstractWe investigated the role of Na+–K+–Cl−co‐transporter isoform 1 (NKCC1) and reversal of Na+/Ca2+exchanger (NCXrev) in glutamate‐mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6–8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na+/Ca2+exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular(), which was followed by a sustainedoverload, NKCC1 phosphorylation, and a NKCC1‐mediated Na+influx. In the presence of a specific AMPA receptor inhibitor 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX), the AMPA/CTZ failed to elicit any changes in. The AMPA/CTZ‐induced sustainedrise led to mitochondrial Ca2+accumulation, release of cytochromecfrom mitochondria, and cell death. The AMPA/CTZ‐elicitedincrease, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCXrev. These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1‐mediated Na+influx. The latter triggers NCXrevand NCXrev‐mediatedoverload and compromises mitochondrial function and cellular viability.