The aim of the present study was to investigate, using in vivo dialysis, the existence of muscarinic and nicotinic receptors controlling acetylcholine release in the cortex of freely behaving rats. Various muscarinic receptor antagonists, including the nonselective blocker atropine, and a variety of M2 drugs (AF-DX116, AF-DX384, AQ-RA 741) potently stimulated, in a concentration-dependent manner, the in vivo release of acetylcholine in the rat cortex. The effects of all these antagonists were long lasting. The nature of these putative muscarinic autoreceptors is likely of the pharmacologically defined M2 subtype on the basis of the high potency of the antagonists of the AF-DX series and the variability and shorter duration of action of the effects of the prototypic M1 blocker, pirenzepine. 4-DAMP, a purported M3 blocker, also potently stimulated in vivo cortical acetylcholine release, but this likely relates to its now established, rather limited selectivity for any given muscarinic receptor subtypes. Peripheral and central injections of nicotine also induced the in vivo release of acetylcholine in the rat cortex, albeit with a lower potency and shorter duration of action than muscarinic antagonists. Interestingly, the combination of a muscarinic antagonist, such as atropine, AF-DX 116, or AF-DX384, in the presence of nicotine, induced tremendous releases of cortical acetylcholine up to 8- to 10-fold over basal values. This is clearly more than a simply additive effect, and it reveals the great capacity of cortical cholinergic nerve terminals to synthesize and release acetylcholine. Optimal pharmacological manipulations of these putative muscarinic and nicotinic autoreceptors could thus be useful in disorders in which the activity of cholinergic inputs is decreased, such as in Alzheimer's disease.