There is evidence that muscarinic receptors of the M2 subtype are presynaptic autoreceptors that modify the release of acetylcholine (ACh) through a negative feedback mechanism. Blocking these receptors by selective antagonists may therefore lead to increased ACh release. This in vivo microdialysis study examined the effects of three M2 antagonists, AF-DX 116, AF-DX 384, and AQ-RA 741, on hippocampal cholinergic neurotransmission. Drug (2, 4, 8, or 16 microM) or vehicle (Ringer's solution) was perfused via a microdialysis probe into the CA1 hippocampal region of conscious male Fischer 344 rats. Levels of ACh and choline were assessed by HPLC-EC. When the dose was expressed in K1 multiples, all drugs (except AQ-RA 741 at the two highest concentrations) were found to be on the same linear dose-response curve. Choline levels were not affected by drug administration. All three compounds elevated ACh levels in a similar K1-normalized dose-response fashion, strongly supporting the concept that the proposed presynaptic mechanism of action is indeed based on the same M2 receptor. Such elevations of ACh may not only improve performance on memory tasks, but may also have therapeutic advantages in conditions of cholinergic hypofunction, such as Alzheimer's disease.