Rapid rejection or immune exclusion of challenge larvae is a well recognised phenomenon in sheep hypersensitised by repeated infection with gastrointestinal nematodes. While mast cells and globule leukocytes (GLs) are typically associated with this rapid rejection response, the exact mechanisms and mediators involved are not known. This study has adapted a recently developed ex vivo tissue explant model to examine in more detail the cells and mediators involved in preventing establishment of Haemonchus contortus L3s in abomasal tissue of sensitised sheep. Hypersensitisation of sheep by repeated larval infection resulted in a significant inhibition of larval establishment in abomasal tissue cultures and the extent of inhibition was dependent on the sensitisation dose. Both mast cells and GLs, but not eosinophils, were increased in abomasal tissues of hypersensitised sheep. Globule leucocyte numbers decreased significantly after 3 h of culture, independent of the addition of L3s. In contrast, mast cell numbers only decreased after addition of L3s to the tissue cultures and this was associated with an increased release of histamine in tissue washes after incubation with L3s. Although there was no significant difference in the number of tissue eosinophils between the groups, there was a marked increase in the eosinophil-specific protein, galectin-14, in tissue washes of the hypersensitised sheep after culture, suggesting eosinophils and their products may play a hitherto unrecognised role in the rapid rejection response. Further studies using specific inhibitors in this ex vivo tissue explant model may delineate the relative role of each cell population and mediator in the rapid rejection process.