The ileal brake is a neural reflex that slows proximal small bowel transit when fat enters the distal small bowel. In rats, ondansetron, a 5-hydroxytryptamine-3 (5-HT3)-receptor antagonist, abolishes the ileal brake. However, the location of this serotonergic pathway is unknown. Of the known enteric sites responsive to 5-hydroxytryptamine (5-HT), only the myenteric neurone is equipped with 5-HT3 receptors and is located on the efferent limb of reflex response. The aim of this study was to test the hypothesis that slowing of intestinal transit by fat may depend on an ondansetron-sensitive serotonergic pathway located on the efferent limb of this reflex response. In a fistulated dog model that compartmentalized the afferent from the efferent limb of the ileal brake response, ondansetron was delivered luminally into the distal (afferent) or proximal (efferent) half of the small bowel to localize the serotonergic pathway. It was found that activating the ileal brake slowed down the proximal intestinal transit to 30% of control values. The ileal brake was abolished when ondansetron was delivered into the proximal but not the distal small bowel. Our data supports the hypothesis that the 5-HT receptors participating in the ileal brake are on the efferent limb of this neural reflex, possibly on myenteric neurones.