The fluorescent probe SBFI was used to monitor the influence of acetylcholine (ACh) on the cytosolic concentration of free Na+ (Na+i) in single mouse pancreatic B-cells. In the presence of 3 mM glucose and 135 mM extracellular Na+, Na+i averaged 16.6 mM. ACh (100 microM) increased Na+i by approximately 80%. This rise was prevented by atropine, a blocker of muscarinic receptors, and by omission of extracellular Na+, but still occurred if the sodium pump was blocked by ouabain. It was unaffected by tetrodotoxin, a blocker of voltage-sensitive Na+ channels, and was not mimicked by depolarization of the cells with high K+. It is concluded that activation of muscarinic receptors increases the membrane permeability to Na+ in pancreatic B-cells.