The influence of calcium on basal and acetylcholine- stimulated pancreatic polypeptide (PP) secretion was investigated in an isolated pancreatico-duodenal preparation and compared to the secretion of glucagon and insulin. The stimulatory effect of 5 mmol/liter calcium on PP release was of the same magnitude as that obtained by 5 mmol/liter arginine or 10 nmol/liter isoproterenol but only one fifth of the PP response to acetylcholine (1 jumol/liter). All stimuli were equipotent with respect to insulin and glucagon release. The acetylcholine (1 jumol/liter) -stimulated PP release was almost identical at calcium concentrations of 1.3 and 6.3 mmol/liter, whereas glucagon release was calcium dependent, with higher responses at high (6.3 mmol/liter) than at normal (1.3 mmol/liter) calcium concentrations. In a calcium-depleted medium, acetylcholine induced a prompt, short-lived, but repeatable PP response, whereas no increase in glucagon or insulin was found. Further, when calcium influx into cells was blocked by excess magnesium (5.0 mmol/liter), the basal and acetylcholine (1 jumol/liter)-stimulated PP secretion was only inhibited by 12% (P = NS) and 42% (2P < 0.05), respectively, whereas glucagon release was inhibited 56% (2P < 0.001) and 76% (2P < 0.01), respectively. It is concluded that the secretion of PP is influenced by calciumions; however, the PP release is much less dependent on extracellular calcium ions than are insulin and glucagon secretions.