ABSTRACT: The studies of the mechanisms by which trauma-hemorrhagic shock leads to gut injury and dysfunction have largely ignored the nonbacterial factors contained within the lumen of the intestine. Yet, there is increasing evidence suggesting that intraluminal pancreatic proteases may be involved in this process. Thus, we tested the hypothesis that pancreatic proteases are necessary for the trauma-hemorrhagic shock-induced gut injury and the production of biologically active mesenteric lymph by determining the extent to which pancreatic duct ligation (PDL) would limit gut injury and mesenteric lymph bioactivity. To assess the effect of PDL on gut injury and dysfunction gut morphology, the mucus layer structure and the gut permeability were measured in the following four groups of male rats subjected to laparotomy (trauma) and hemorrhagic shock (pressure, 30 mmHg for 90 min): (1) rats subjected to trauma plus sham-shock (T/SS), (2) T/SS rats undergoing PDL (T/SS + PDL), (3) rats subjected to trauma and hemorrhagic shock (T/HS), and (4) rats subjected to T/HS + PDL. The ability of mesenteric lymph from these four rat groups to kill endothelial cells and activate neutrophils was tested in vitro. The PDL did not affect any of the parameters studied because there were no differences between the T/SS and the T/SS + PDL groups. However, PDL protected the gut from injury and dysfunction because PDL significantly abrogated T/HS-induced mucosal villous injury, loss of the intestinal mucus layer, and gut permeability. Likewise, PDL totally reversed the endothelial cell cytotoxic activity of T/HS lymph and reduced the ability of T/HS lymph to prime naive neutrophils for an augmented respiratory burst. Thus, it seems that intraluminal pancreatic proteases are necessary for the T/HS-induced gut injury and the production of bioactive mesenteric lymph.