Dietary protocols that increase serum levels of ketones, such as calorie restriction and the ketogenic diet, offer robust protection against a multitude of acute and chronic neurological diseases. The underlying mechanisms, however, remain unclear. Previous studies have suggested that the ketogenic diet may reduce free radical levels in the brain. Thus, one possibility is that ketones may mediate neuroprotection through antioxidant activity. In the present study, we examined the effects of the ketones beta-hydroxybutyrate and acetoacetate on acutely dissociated rat neocortical neurons subjected to glutamate excitotoxicity using cellular electrophysiological and single-cell fluorescence imaging techniques. Further, we explored the effects of ketones on acutely isolated mitochondria exposed to high levels of calcium. A combination of beta-hydroxybutyrate and acetoacetate (1 mM each) decreased neuronal death and prevented changes in neuronal membrane properties induced by 10 microM glutamate. Ketones also significantly decreased mitochondrial production of reactive oxygen species and the associated excitotoxic changes by increasing NADH oxidation in the mitochondrial respiratory chain, but did not affect levels of the endogenous antioxidant glutathione. In conclusion, we demonstrate that ketones reduce glutamate-induced free radical formation by increasing the NAD+/NADH ratio and enhancing mitochondrial respiration in neocortical neurons. This mechanism may, in part, contribute to the neuroprotective activity of ketones by restoring normal bioenergetic function in the face of oxidative stress.