In the present study, we investigated the site in the host where protective gut immunity to Fasciola hepatica is induced and expressed, following the infection route of the parasite. Expression of protection was studied in ex vivo gut segments with intact blood and lymph supply that were prepared at different locations along the entire length of the small and large intestine. Four weeks after oral infection, significant protection was detected in the duodenum, upper jejunum, midjejunum, and ileum. Protection at the gut level was expressed as early as 2 wk after oral priming and waned after 27 wk. The possibility that the gut wall plays a role in age-related protection was excluded. The effect of newly excysted juveniles (NEJ) penetrating the gut on the induction of protection was studied by recovering or killing the NEJs of the primary infection immediately after gut migration. Results showed that protection was low (13.9-19.8%). However, when gut migration was by-passed and NEJs of the primary infection were injected into the peritoneal cavity or between the liver lobes, high levels of protection at the gut level were detected (76.5-87.4%). The results indicate that protection expressed at the gut level is induced by the parasite at a young stage, during migration through the peritoneal cavity, or liver, or both and not during penetration of the gut.