The tritiated muscarinic cholinergic antagonist quinuclidinyl benzilate, [3H]QNB, was used as a direct probe for the detection and characterization of muscarinic cholinergic receptors associated with the particulate fraction of isolated and purified rat gastric muscosal parietal cells. Specific binding is saturable (Bmax = 55 fmol/mg protein, KD = 0.78 nM), shows a single population of binding sites, and has appropriate pharmacological specificity. Nanomolar concentrations of muscarinic cholinergic antagonists, such as atropine and scopolamine, inhibit [3H]QNB binding by 50%, whereas micromolar concentrations are needed for agonists, such as acetylcholine, oxotremorine, and carbamylcholine. Binding is also stereoselective as shown by the more than 1,000-fold difference in inhibitory potencies of the stereoisomers of benzetimide. Noncholinergic agents, including pentagastrin, histamine, and the H2-receptor antagonists cimetidine and metiamide, have little or no effect on [3H]QNB binding at concentrations of 100 microM. These data support the existence of specific parietal cell muscarinic cholinergic receptors with which the secretagogue acetylcholine may directly interact to initiate gastric acid secretion.