Long-term use of antiepileptic drugs is common in the treatment of epilepsy. Clinical reports exist of cognitive impairment attributed to antiepileptic drugs. Hence, this study evaluates the effect of chronic administration of one antiepileptic drug, ethosuximide, on spatial and fear learning and memory in nonepileptic rats. High performance liquid chromatography with electrochemical detection was used for quantification of glutamate, glycine, taurine, gamma-aminobutyric acid, dopamine, and serotonin in the frontal cortex and hippocampus to elucidate the neurobiological basis of the effect of ethosuximide on learning and memory. We found that 21 days of ethosuximide treatment produced negative effects on fear memory (passive avoidance) at all doses (100, 200 and 250 mg/kg body weight), but had no effect on spatial learning (T-maze). Fear memory impairment was associated with decreased hippocampal dopamine levels. Ethosuximide (at all doses) had a minimal effect on the GABAergic and glutamatergic systems in all brain regions studied, with the exception of elevated levels of gamma-aminobutyric acid in the frontal cortex with the 250 mg/kg body weight dose. We have shown that long-term administration of ethosuximide adversely affects fear memory, but does not affect spatial learning and memory.