In previous investigations, we have demonstrated that cholinesterase inhibitors such as physostigmine (PHY) and heptylphysostigmine (HEP) elicit a significant and simultaneous increase in acetylcholine (ACh) and norepinephrine (NE) levels in the rat cortex. This effect is enhanced by idazoxan, a selective alpha-2 antagonist. These data suggest that a combination of cholinergic and adrenergic drug may improve the pharmacological effect of the cholinesterase inhibitor on cortical neurotransmitters such as ACh-NE. In order to obtain additional information on cortical cortical neurotransmitter interaction, we tested, in the cerebral cortex of the rat, the effect of PHY and HEP in animals pretreated with clonidine (CLO), a selective alpha-2 agonist, on ACh, NE, dopamine and 5-hydroxytryptamine) extracellular levels. We detected no effect of systemic or intracortical CLO administration of ACh levels, but NE, dopamine and 5-hydroxytryptamine levels were all decreased. Systemic coadministration of CLO and PHY significantly elevated ACh levels and decreased NE, dopamine and 5-hydroxytryptamine levels. Systemic coadministration of CLO and HEP produced a significant elevation in ACh levels. Comparison between the two treatment combinations shows that, although CLO coadministration reduces the effect of PHY on ACh levels, HEP administered to animals pretreated with CLO produces a stronger effect than HEP alone. A possible explanation for this difference is the variation in duration of the two drugs on ACh elevation and muscarinic receptor desensitization. As a result of the alpha-2 agonist cholinesterase inhibitor coadministration, our data suggest that such a combination does not represent an advantage as a therapeutical alternative for treatment of cognitive impairment in Alzheimer disease patients.