Few previous studies have discussed the changes in serotonin receptor activity in the small intestine of diabetic animals. Therefore, we examined serotonin content in duodenal tissue and dose-dependent effects of serotonin agonists and antagonists on the motor activity of ex vivo vascularly perfused duodenum of streptozotocin (STZ)-diabetic rats. Serotonin content was significantly increased in enterochromaffin cells but not altered in serotonin-containing neurons in STZ-diabetic rats. Motor activity assessed by frequency, amplitude, and percent motility index per 10 min of pressure waves was reduced in the duodenum of diabetic rats, and this reduction was reversed by insulin treatment. Serotonin dose dependently increased the motor activity in control rat duodenum but only a higher concentration of serotonin increased the motor activity in diabetic rats. The 5-hydroxytryptamine (5-HT) receptor subtype 4 (5-HT₄) antagonist SB-204070 dose dependently reduced motor activity in both control and diabetic rats, whereas the 5-HT₃ receptor antagonist azasetron, even at a higher concentration, failed to affect motor activity in diabetic rat duodenum but dose dependently reduced motor activity in control rat duodenum. These results suggest that 5-HT₃ receptor activity was impaired but 5-HT₄ receptor activity was intact in STZ-diabetic rat duodenum. Such an impairment of 5-HT₃ receptor activity may induce the motility disturbance in the small intestine of diabetes mellitus.