We have previously shown that serotonin type-3 (5-HT3) receptors mediate cholecystokinin (CCK)-induced satiation and that this effect is dependent on post-oropharyngeal feedback. However, the independent contributions of gastric and intestinal feedback in 5-HT3 receptor mediation of suppression of food intake by CCK have not been determined. Using a sham feeding preparation combined with intraduodenal sucrose infusion, we show that blockade of 5-HT3 receptors by ondansetron (1mg/kg; IP) had no effect on suppression of sham feeding by intraduodenal 15% sucrose infusion (4ml/10min), CCK (2µg/kg; IP) administration, or the combination of the two treatments. In separate experiments consisting of either sham feeding rats that received gastric distension using a balloon or real feeding rats whose stomachs were distended using gastric loads of saline following the occlusion of the pylorus, we tested the hypothesis that gastric feedback signals are necessary for activation of 5-HT3 receptors. Ondansetron significantly attenuated suppression of sham sucrose intake following a10cc gastric balloon distension (30. 5 ± 2.2 vs. 20.2 ± 2.2ml, respectively) and gastric distention combined with CCK (21.9 ± 1.4 vs. 12.0 ± 1.7, respectively). When intestinal feedback was eliminated in a real feeding paradigm by closing the pylorus using a cuff preparation, ondansetron attenuated suppression of sucrose intake produced by a 10ml saline gastric load (6.8 ± 0.7 vs. 4.2 ± 0.4ml, respectively). Finally, when CCK (1µg/kg) was administered in combination with a 5ml saline gastric load in a real feeding preparation, ondansetron significantly attenuated suppression of sucrose intake by CCK (9.0 ± 0.9 vs. 6.3 ± 0.5ml, respectively), as well as the enhanced suppression of intake by CCK plus gastric load (6.9 ± 0.6 vs. 4.6 ± 0.5ml, respectively). These findings demonstrate that CCK-induced activation of 5-HT3 receptors requires gastric, but not intestinal feedback.