Hypothalamic obese rats, obtained by neonatal treatment with monosodium l-glutamate (MSG), are hyperinsulinemic, and secrete more insulin than lean ones do when stimulated by glucose, while acetylcholine insulinotropic effect decreases. The effect of acetylcholine on glucose-induced insulin secretion is attributed to muscarinic receptors of pancreatic beta cells, mainly to M3 subtype. However, it has been observed that activation of M2 or M4 subtypes causes inhibition of glucose-induced insulin secretion in insulin secreting cell line. Insulin secretion was measured, stimulated by glucose in the presence of acetylcholine plus methoctramine, a muscarinic M2 antagonist, on pancreatic islets isolated from MSG-obese and lean rats to investigate whether impairment of acetylcholine insulinotropic effect on pancreatic islets from MSG-obese rats has any relationship with muscarinic M2 receptor function in beta cells. Insulin secretion stimulated by 8.3 mM glucose was higher in islets from obese rats than from lean ones. Insulinotropic effect of acetylcholine was reported in islets of both animals, albeit less than in obese ones. Blockage of muscarinic M2 receptor, using methoctramine at 1; 5 and 10 μM, increased acetylcholine secretory response in islets of obese rats, while no effect has been observed in lean ones. Results demonstrate that muscarinic M2 receptors are functioning in pancreatic islets of MSG-obese rats. The inhibitory action of muscarinic M2 receptor may be a mechanism by which acetylcholine discloses weak insulinotropic effect in MSG-obese rats.