Cholinergic agents are potent modulators of insulin release that act via muscarinic receptors. We now investigated the muscarinic receptor subtype present in rat pancreatic islets in binding and functional studies. Binding of 5 nM [3H]N-methylscopolamine ([3H]NMS) was half maximal at 30 min. At 60 min, the maximal total binding was 1.29% and the non-specific binding (presence of 100 microM atropine) was 0.18% of the total radioactivity per 10 micrograms islet protein. Unlabelled atropine inhibited [3H]NMS binding with an IC50 of ca. 30 nM. The rank order of antagonist high-affinity binding was atropine greater than sila-hexocyclium methyl sulfate (SiHC; M1 greater than M3 greater than M2) greater than pirenzepine (M1 greater than M2 approximately M3) = methoctramine (M2 greater than M1 greater than M3). The high-affinity Kds were 8.5, 56, 1300 and 1300 nM, respectively. The high affinity Kd of the muscarinic receptor agonist, arecaidine propargyl ester (APE), was 8.1 nM. The EC50 for the biological effects of APE on insulin and glucagon secretion was 3.2 and 2.3 nM. The rank order for the high-affinity biological effects of antagonists (inhibition of APE-mediated insulin/glucagon release) was almost the same as for binding. The data indicate that rat pancreatic islets contain neither an M1 subtype (high-affinity for pirenzepine) nor an M2 subtype (high-affinity for methoctramine) receptor. However, the data evidence an M3 receptor subtype, since SiHC in the absence of the M1 receptor subtype shows a relatively high affinity to the receptors in rat pancreatic islets.