The learning and memory in adult, male Wistar rats were assessed using the T-maze and passive avoidance tests after chronic administration of phenobarbital (PB) at 5, 15, 30, 60, or 75 mg/kg intraperitoneally (IP) for 21 days. The PB levels in plasma, the acetylcholine esterase (AChE) activity in the motor cortex, pyriform cortex, olfactory bulb, striatum, septum, and hippocampus and the levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) levels in the hippocampus were measured. There was no significant change in learning and memory, AChE activity, or monoamine levels at plasma PB levels of 1.5, 6.0, 9.0, and 25 micrograms/ml (corresponding to doses of 5, 15, 30, or 60 mg/kg PB, respectively). However, at a plasma level of 55 micrograms/ml (75 mg/kg), PB caused impairment in learning and memory. It was associated with an increase in AChE activity and 5-HT levels in the hippocampus. The results indicate that chronic PB administration may not be linked to impaired learning and memory functions at doses used in anticonvulsant therapy.