Trypsin-treated, cell-free filtrates derived from enterotoxigenic Escherichia coli, strain H197 (O78:H11), exhibited a fourfold or greater increase in heat-labile vascular permeability factor activity and a 10-fold or greater increase in the ability to stimulate secretion of growth hormone by cultured rat pituitary cells. In contrast, trypsin-treated filtrates were not different from untreated filtrates in their ability to elicit a secretory response in ligated rabbit intestinal loops. However, incubation of culture filtrate in ligated intestinal loops, or with rabbit intestinal fluid (in vitro), resulted in at least a twofold increase in permeability factor that did not occur in the presence of trypsin inhibitor or with heat-inactivated intestinal fluid. Moreover, trypsin inhibitor could reduce the secretory response to culture filtrate. These findings suggest that activation of heat-labile E. coli enterotoxin by host enzymes may play a role in the development of a full pathogenic effect.