Aim: To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats. Methods: Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters. Results: Our results showed that huperzine A (0.25, 0.5, and 0.75 μmol/kg, po) dose-depen-dently elevated extracellular acetylcholine (Ach) levels in the medial prefrontal cortex (Mpfc) and hippocampus. Oral administration of donepezil (5.4 μmol/kg) or rivastigmine (1 μmol/kg) also elicited significant increases in Ach in the Mpfc and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of Ach at the same dose. The marked elevation of Ach after oral administration of huperzine A (0.5 μmol/kg) and donepezil (5.4 μmol/kg) was associated with a significantly increased release of dopamine (DA) in the Mpfc or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the Mpfc and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase Ach levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 μmol/kg, once per day for 30 d. Conclusion: The present findings showed that, in molar terms, huperzine A had similar potency on increasing Mpfc Ach and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing.