Prejunctional affinity constants of the cardioselective muscarine receptor antagonist AF-DX 116 (11-[(2-[(diethyl-amino)methyl]-1-piperidinyl)acetyl]-5,11-dihydro-6 H-pyrido [2,3-b] [1,4] benzodiazepine-6-one) were determined for muscarine autoreceptors on cholinergic nerves of the guinea-pig ileum and for heteroreceptors on noradrenergic nerves of the rat heart and guinea-pig iris. AF-DX 116 antagonized with low affinity the muscarinic inhibition induced by arecaidine propargyl ester of the stimulation-evoked [3H]acetylcholine overflow (pA2 6.74) from the guinea-pig ileum. In contrast, AF-DX 116 was more potent in antagonizing the methacholine-induced inhibition of the stimulation-evoked [3H]noradrenaline overflow from rat heart (pA2 7.29) or guinea-pig iris (pA2 7.57). The data confirm previously reported differences between prejunctional muscarine heteroreceptors in the rat heart which belong to the cardiac subtype (M2 alpha or M2) and autoreceptors in the guinea-pig ileum that cannot be distinguished from the ileal subtype (M2 beta) or M3).