The vago-vagal reflex plays an important role in mediating pancreatic secretion evoked by cholecystokinin and non-cholecystokinin-dependent luminal factors. We hypothesize that the vago-vagal reflex mediating pancreatic secretion in the rat is under central control and regulated by cholinergic pathways in the hypothalamus. To test this hypothesis, we demonstrated that chronic decerebration decreased basal pancreatic enzyme secretion from 318 ± 12 to 233 ± 9 mg h - 1 and reduced the net increase in pancreatic secretion stimulated by intraduodenal infusion of 5% peptone and hypertonic NaCl by 54% and 45%, respectively. Intracerebroventricular administration of methscopolamine (MSCP, 50 nmol/5 µ l), a blood brain barrier impermeant cholinergic muscarinic receptor antagonist, evoked similar results to those achieved by chronic decerebration. To localize the sites of action, we demonstrated that microinjection of MSCP (20 nmol) into the lateral hypothalamic nucleus and paraventricular nucleus resulted in inhibition of basal pancreatic secretion and luminally stimulated pancreatic protein secretion by 48% and 52%, respectively. Intracerebroventricular injection of hemicholinium-3 at doses known to deplete the endogenous ACh store produced similar inhibitory results. In addition, microinjection of ACh (5 pmol) or the muscarinic M1 receptor agonist McN-A-343 (30 ng) into the lateral hypothalamic nucleus increased pancreatic secretion over basal by 46% and 40%, respectively. Selectively lesion of lateral septal (LS) cholinergic neurons decreased basal pancreatic secretion and inhibited peptone-induced pancreatic secretion by 30%. Destroying of the lateral parabrachial nucleus (LPBN) produced a 44% inhibition of peptone- induced pancreatic section. Finally, microinjection of glutamate into the LS or the LPBN stimulated vagal pancreatic efferent nerve firings from basal 0 ± 0.5 to 4.5 ± 0.5 and 14 ± 2 impulses 30 s-1, respectively, and caused 50% and 84% increases of pancreatic protein output over basal, effects were eliminated by PVN administration of MSCP. These observations suggest that LS and LPBN cholinergic neurons regulate pancreatic secretions. LPBN cholinergic input to the hypothalamus plays a major role in the modulation of vagal pancreatic efferent nerve activity and pancreatic secretion evoked by vago-vagal reflex.