1. The role of ghrelin in the regulation of pancreatic protein secretion was investigated in vivo using anaesthetized rats with pancreatic ductal cannulas, and in isolated pancreatic acinar cells and pancreatic lobules in vitro. 2. In vivo, pancreatic protein output stimulated by CCK-8 (400 pmol kg-1 h-1) was dose-dependently inhibited by continuous ghrelin infusion (1.2 and 12 nmol kg-1 h-1) by 45 ± 8 and 84 ± 7%, respectively. 3. In rats with acute subdiaphragmatic vagotomy, ghrelin (12 nmol kg-1 h-1) significantly inhibited CCK-stimulated pancreatic protein secretion by 75 ± 18%. 4. Infusion of ghrelin (12 nmol kg-1 h-1) abolished pancreatic protein secretion caused by the central vagal stimulant 2-deoxy-d-glucose (75 mg kg-1), whereas bethanechol-stimulated pancreatic protein output was inhibited by only 59 ± 7%. 5. In vitro, ghrelin (10-11-10-7 m) produced no change in basal amylase release from dispersed, purified acinar cells. Co-incubation of ghrelin (10-11-10-7 m) with CCK-8 (10-10 m) demonstrated no inhibition of CCK-stimulated amylase release from dispersed acini. In contrast, ghrelin (10-9-10-7 m) dose-dependently inhibited amylase release from pancreatic lobules exposed to 75 mm potassium. 6. Our results show that (1) ghrelin is a potent inhibitor of pancreatic exocrine secretion in anaesthetized rats in vivo and in pancreatic lobules in vitro; and (2) the actions of ghrelin are indirect and may be exerted at the level of intrapancreatic neurons.