Shock and multiple organ failure (MOF) remain primary causes of late stage morbidity and mortality in victims of trauma. During shock the intestine is subject to extensive cell death and is the source of inflammatory factors that cause multi-organ failure. We showed previously that ischemic, but not non-ischemic, small intestines and pancreatic protease digested homogenates of normal small intestine can generate cytotoxic factors capable of killing naive cells within minutes. Using chloroform/methanol separation of rat small intestine homogenates into lipid fractions and aqueous and sedimented protein fractions and measuring cell death caused by those fractions, we found that the cytotoxic factors are lipid in nature. Re-combining the lipid fraction with protein fractions prevented cell death except when homogenates were protease digested. Using a fluorescent substrate, we found high levels of lipase activity in intestinal homogenates and cytotoxic levels of free fatty acids. Addition of albumin, a lipid-binding protein, prevented cell death, unless the albumin was previously digested with protease. Homogenization of intestinal wall in the presence of the lipase inhibitor Orlistat prevented cell death after protease digestion. In vivo, Orlistat plus the protease inhibitor, aprotinin, administered to the intestinal lumen significantly improved survival time compared to saline in a splanchnic arterial occlusion model of shock. These results indicate that major cytotoxic mediators derived from an intestine under in-vitro conditions are free fatty acids. Breakdown of free fatty acid-binding proteins by proteases causes release of free fatty acids to act as powerful cytotoxic mediators.