1 Relaxation responses of the rat isolated duodenum to the putative M1 muscarinic receptor agonist, McN-A-343, were examined to determine whether the response was due to the release of known non-adrenergic, non-cholinergic relaxant neurotransmitters and to establish the involvement of M1 muscarinic receptors. 2 The role of ATP was examined with the P2 receptor antagonist, suramin, which at 30 μm antagonized the relaxant responses to α,β-methylene ATP. The same dose, however, failed to inhibit the relaxation by McN-A-343. 3 The role of nitric oxide (NO) was examined with the NO synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME; 100 μm), which failed to inhibit the responses to McN-A-343. As NO mediates relaxation of the duodenum via cGMP generation through guanylyl cyclase, whether the relaxation by McN-A-343 was also via cGMP was examined with the guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). The relaxation responses to the NO donor, S-nitroso-N-acetyl penicillamine, were inhibited in the presence of ODQ (3 μm), but not those by McN-A-343. 4 Release of γ-aminobutyric acid (GABA) was examined with the GABAA receptor antagonist, bicuculline (10 μm), which shifted the concentration–response curves for the relaxation of the duodenum by GABA to the right. There was a similar degree of shift in the concentration–response curve for McN-A-343 by bicuculline indicating that release of GABA from enteric neurones of the duodenum could explain the relaxation response to McN-A-343. 5 To test whether the muscarinic receptors mediating the relaxation of the duodenum were of the M1 subtype, the susceptibility to the selective competitive antagonist, pirenzepine and the selective muscarinic toxin from green mamba, MT7, was examined. Pirenzepine (1 μm) shifted the concentration–response for McN-A-343 to the right in a parallel fashion with a dose ratio of 33.3 ± 20.2. This yielded a pA2 value of 7.5, which concords with those for other responses reputed to be mediated via M1 muscarinic receptors. The toxin MT7 was used as an irreversible antagonist and following incubation with the duodenum was washed from the bath. An incubation time of 30 min with 100 nm of MT7 caused a significant parallel shift in the concentration–response to McN-A-343 confirming the involvement of M1 muscarinic receptors. 6 This study has confirmed that McN-A-343 relaxes the rat duodenum via muscarinic receptors of the M1 subtype and that these receptors are probably located on enteric neurones from which their stimulation releases GABA.