Acetylcholine (ACh), the major parasympathetic neurotransmitter, is released by intrapancreatic nerve endings during the preabsorptive and absorptive phases of feeding. In β-cells, ACh binds to muscarinic M₃ receptors and exerts complex effects, which culminate in an increase of glucose (nutrient)-induced insulin secretion. Activation of PLC generates diacylglycerol. Activation of PLA₂ produces arachidonic acid and lysophosphatidylcholine. These phospholipid-derived messengers, particularly diacylglycerol, activate PKC, thereby increasing the efficiency of free cytosolic Ca²⁺ concentration ([Ca²⁺]_c) on exocytosis of insulin granules. IP3, also produced by PLC, causes a rapid elevation of [Ca²⁺]_c by mobilizing Ca²⁺ from the endoplasmic reticulum; the resulting fall in Ca²⁺ in the organelle produces a small capacitative Ca²⁺ entry. ACh also depolarizes the plasma membrane of β-cells by a Na⁺- dependent mechanism. When the plasma membrane is already depolarized by secretagogues such as glucose, this additional depolarization induces a sustained increase in [Ca²⁺]_c. Surprisingly, ACh can also inhibit voltage-dependent Ca²⁺ channels and stimulate Ca²⁺ efflux when [Ca²⁺]_c is elevated. However, under physiological conditions, the net effect of ACh on [Ca²⁺]_c is always positive. The insulinotropic effect of ACh results from two mechanisms: one involves a rise in [Ca²⁺]_c and the other involves a marked, PKC-mediated increase in the efficiency of Ca²⁺ on exocytosis. The paper also discusses the mechanisms explaining the glucose dependence of the effects of ACh on insulin release.