To investigate the neural regulation of pancreatic polypeptide (PP) secretion during hypoglycemia in the rat, insulin was administered to chronically cannulated rats, and plasma PP responses were compared between saline-treated animals and animals pretreated with a ganglionic blocking agent (hexamethonium), a muscarinic antagonist (atropine), combined alpha- and beta-adrenergic receptor blockade (propranolol + tolazoline), or combined adrenergic blockade + atropine. PP was measured using a new RIA which selectively detects PP in rat plasma. In control rats (n = 10), plasma PP increased from a baseline level of 30 +/- 3 pg/ml to 271 +/- 41 pg/ml during hypoglycemia (plasma glucose = 29 +/- 2 mg/dl) (delta PP = +241 +/- 42 pg/ml, P less than 0.0005), demonstrating that in rats, as in other species, insulin-induced hypoglycemia is a potent stimulus for PP release. PP only increased by 31 +/- 10 pg/ml during similar hypoglycemia in 7 hexamethonium-treated rats (P less than 0.01 vs. control animals). Thus, at least 90% of the PP response to hypoglycemia is neurally mediated. The plasma PP response to hypoglycemia was +85 +/- 24 pg/ml in atropine-treated rats (P 0.01 vs. control rats), suggesting that approximately 65% of the PP response is mediated via muscarinic acetylcholine receptors on the islet F cell. The PP response to hypoglycemia in rats with combined adrenergic blockade (delta = +168 +/- 32 pg/ml) was slightly, but not significantly smaller than that in control rats. The combination of combined blockade + atropine resulted in a PP response (delta = +26 +/- 7 pg/ml) to hypoglycemia that was similar to that in hexamethonium-treated rats (P less than 0.01 vs. control rats). These results suggest: 1) The PP response to hypoglycemia is predominantly the result of muscarinic, cholinergic activation. 2) There is a minor adrenergic contribution to the response. 3) The plasma PP response may be useful as an index of autonomic neural input to the islet during hypoglycemia.