Neuronal apoptosis has been shown to be associated with the development of tolerance to morphine. In the present study, we investigated the effect of intracerebroventricular (icv) administration of an inhibitor of glutamate release, riluzole, on morphine-induced apoptosis in the rat cerebral cortex. Various groups of rats received either morphine (intraperitoneally, ip) and vehicle (icv) or morphine (ip) and different doses of riluzole (icv) once per day for 8 days. An in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method was used as an apoptosis assay. Levels of the anti-apoptotic factors Bcl-2 and HSP70 and the pro-apoptotic agent caspase-3 were evaluated by immunoblotting. The glutamate concentration in the cerebral cortex was measured by high performance liquid chromatography (HPLC). The results showed that icv administration of riluzole decreased the number of apoptotic cells in the cerebral cortex compared with the control group, which was treated with morphine (ip) and 1% Tween 80 in 0.9% normal saline (icv). The levels of the anti-apoptotic proteins Bcl-2 and HSP70 were higher in the riluzole groups than in the control. Furthermore, co-administration of riluzole with morphine significantly decreased caspase-3 protein levels and glutamate content of the cerebral cortex compared with the control. In conclusion, we found that icv administration of riluzole attenuates morphine-induced apoptosis in the cerebral cortex after the development of morphine tolerance.