Cholecystokinin and serotonin are released from the gastrointestinal tract in response to the products of digestion and play critical roles in mediating pancreatic secretion via vago-vagal reflex pathways. This study was designed to investigate the effects of activation of cholecystokinin CCK1 and serotonin (5-hydroxytryptamine, 5-HT) 5-HT3 receptors on pancreatic vagal afferent discharge and to determine whether there is an interaction between these receptors. Male Sprague Dawley rats anaesthetised with isoflurane (1.5%/100% O2) were used in all experiments. The effects of systemic administration of cholecystokinin and the serotonin 5-HT3 receptor agonist phenylbiguanide on pancreatic vagal afferent discharge were recorded before and after administration of cholecystokinin CCK1 and serotonin 5-HT3 receptor antagonists. Cholecystokinin (0.1–10 μg/kg, i.v.) and phenylbiguanide (1 and 10 μg/kg, i.v.) increased pancreatic vagal afferent discharge dose-dependently. Cholecystokinin CCK1 receptor antagonists, lorglumide (10 mg/kg, i.v.) and devazepide (0.5 mg/kg, i.v.), reduced cholecystokinin- and phenylbiguanide-induced increases in pancreatic vagal afferent discharge significantly (n = 5, P < 0.05). On the other hand, serotonin 5-HT3 receptor blockade with granisetron (1 mg/kg, i.v.) or MDL72222 ([(1S,5R)-8-methyl-8-azabicyclo[3.2.1]octan-3-yl] 3,5-dichlorobenzoate; 0.1 mg/kg, i.v.) inhibited the pancreatic vagal afferent discharge responses to phenylbiguanide but not those to cholecystokinin. This study has confirmed that cholecystokinin and phenylbiguanide activate pancreatic vagal afferent discharge via activation of cholecystokinin CCK1 and serotonin 5-HT3 receptors, respectively. In addition, it has demonstrated that (i) the serotonin 5-HT3 agonist phenylbiguanide acts partly via an interaction with cholecystokinin CCK1 receptors, and (ii) the actions of cholecystokinin are not dependent on serotonin 5-HT3 receptor activation.