Strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in farm animals and humans during the past decade. Our laboratory has purified and characterized this enterotoxin as a single polypeptide (M(r), approximately 20,000). Recently, we used PCR to clone and sequence the enterotoxin gene from B. fragilis and showed that it exhibits significant homology with extracellular metalloproteases. Further studies showed that the purified enterotoxin has protease activity. To further characterize the role of this enterotoxin in diarrheal disease, we studied the histological and pathological effects of highly purified B. fragilis enterotoxin in lamb, rabbit, and rat ligated intestinal loops. When the enterotoxin was injected into ligated ileal and colonic loops, there was significant tissue damage and subsequent fluid accumulation. The fluid response in the ileum was greater in lambs than in rabbits and rats, whereas the fluid response in the colon was greater in rabbits than in lambs and rats. Analysis of the intestinal fluid elicited by the enterotoxin revealed an accumulation of chloride and sodium as well as albumin and total protein. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to the chelated enterotoxin. Our results indicate that the enterotoxin elicits a significant fluid response subsequent to tissue damage in the small and large intestine. These data further support the idea that this enterotoxin is an important virulence factor in B. fragilis-associated diarrhea.