Phenytoin (DPH) is a widely used anticonvulsant drug but a conclusive mode of action is not yet clear. This study was undertaken to assess the effects of chronic administration of DPH on monoamine levels. DPH (50 mg/kg body weight) was administered to adult male Wistar rats by intraperitoneal injections for 45 days and the regional brain levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were assayed using high performance liquid chromatographic (HPLC) method. The experimental rats revealed no behavioral deficits of any kind nor body and brain weight deficits were observed. Increased NE levels were observed after DPH administration in motor cortex (P < 0.05), striatum-accumbens (P < 0.01) and hippocampus (P < 0.01), whereas, NE level was decreased in brain stem (P < 0.05). DA levels were increased in striatum-accumbens (P < 0.05), hypothalamus (P < 0.001) and cerebellum (P < 0.001) but decreased in brainstem (P < 0.01). In DPH treated rats, 5-HT levels were increased in motor cortex (P < 0.001) but decreased in cerebellum (P < 0.001) when compared to control group of rats. The present study suggest that chronic administration of DPH induces alterations in monoamine levels in specific brain regions. DPH seems to mediate its anticonvulsant action by selectively altering the monoamine levels in different brain regions.