Several gastrointestinal stimuli, including some intestinal nutrients, exert their satiating effect via activation of serotonin type-3 (5-HT₃) receptors. The presence of lipids in the small intestine potently suppresses food intake, however whether 5-HT₃ receptors play a role in this response has not been directly examined. Therefore, using the selective 5-HT₃ receptor antagonist ondansetron, we tested the hypothesis that duodenal infusion of lipid suppresses intake of both sucrose solution and chow through 5-HT₃ receptor activation. Rats duodenally infused with 72, and 130mM Intralipid suppressed 1 hr 15% sucrose intake by 33, and 67%, respectively. Suppression of sucrose intake by 72mM Intralipid was significantly attenuated by ondansetron at all doses tested (0.5, 1.0, 2.0, and 5.0 mg/kg; i.p.), whereas the lowest effective dose of ondansetron to attenuate suppression of intake by 130mM Intralipid was 1.0 mg/kg. Furthermore, infusion of 130mM Intralipid suppressed 1 and 4hr chow intake by 35, and 20%, respectively. Ondansetron administered as low as 0.5 mg/kg significantly attenuated 1hr Intralipid-induced suppression of chow intake and completely reversed the suppression by 4hr. Ondansetron alone did not alter sucrose or chow intake compared with vehicle injection at any time. Finally, to test whether Intralipid-induced neuronal activation of the dorsal vagal complex is mediated by 5-HT₃ receptors, Fos-like immunoreactivity (Fos-LI) was quantified in ondansetron-pretreated rats following intestinal lipid infusion. Ondansetron (1mg/kg) significantly attenuated duodenal intralipid-induced Fos-LI in the dorsal hindbrain. These data support the hypothesis that 5-HT₃ receptors mediate both satiation, as well as hindbrain neuronal responses evoked by intestinal lipids.