We have used a reverse hemolytic plaque assay and frequency distribution of immunoplaque areas to analyze the effect of carbachol (CCh, 100 nM), on insulin secretion by single pancreatic beta-cells. The CCh effect was strongly dependent on the extracellular glucose concentration. Compared with the respective controls in each condition, when glucose was omitted from the incubation medium, CCh induced a 85% increase in the insulin secretion index. In 5.6 mM glucose, CCh induced a 100% increase in the insulin secretion index and this effect was characterized by (1) amplification of the response to glucose, and (2) recruitment of previously silent cells to secretory activity. However, at high glucose concentrations (20.6 mM), the insulin secretion index decreased 49%. CCh effects were blocked by atropine (1 microM). CCh effects were not uniform among beta-cells. The functional subpopulation of beta-cells with the highest secretion rate was preferentially affected by the muscarinic agonist. The specific sodium channel blocker tetrodotoxin prevented CCh-stimulated insulin secretion in basal media, suggesting that voltage-dependent sodium channels are involved in CCh stimulation-secretion coupling in single beta-cells.