Acetylcholine has a variety of actions on hippocampal neurons, possibly through the activation of different muscarinic receptor subtypes. We have used several competitive muscarinic antagonists and the hippocampal slice preparation to test the hypothesis that a particular muscarinic response can be ascribed to a specific subtype. Initially we performed experiments to determine the relative ability of 3 antagonists to inhibit the cholinergic slow EPSP. Dose-response curves were constructed, and the IC50s determined for 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), pirenzepine (PRZ) and 11-2[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11,-dihydro- 6H-pyrido[2,3-b] [1,4]benzodiazepin-6-one (AF-DX 116) were 72 nM, 385 nM, and 24 microM, respectively. While this pattern of antagonism argues against the contribution of the M2 receptor subtype, which would be expected to be relatively more sensitive to antagonism by AF-DX 116, this evidence does not adequately discriminate between other muscarinic receptor subtypes. We therefore performed studies designed to determine the antagonist binding affinities that can be diagnostic for determining different pharmacologically defined receptor subtypes. Using Schild plot analysis we examined the carbachol-induced block of the potassium (K)-dependent leak current, which is the muscarinic response thought to underlie the generation of the synaptically evoked slow excitatory postsynaptic potential (EPSP) in hippocampal neurons. Estimated affinity constants (KBs) for 4-DAMP, PRZ and AF-DX 116 were 7.9, 278 and 1364 nM, respectively. These values are in good quantitative agreement with data from binding studies and strongly suggest a physiological role for the pharmacologically defined 'M3' receptor in the block of the leak conductance and, hence, in the slow EPSP.(ABSTRACT TRUNCATED AT 250 WORDS)