Infant pigs orally exposed to strains of Escherichia coli which produce heat-labile enterotoxin (LT) in vitro, developed diarrhea, and, with the use of the sensitive adrenal cell tissue culture assay for LT, fecal and intestinal materials from these infected animals were shown to contain LT. The rabbit-ligated jejunal loop assay did not detect the LT present in these materials. No enterotoxin was detected by tissue culture assays of such samples from pigs exposed to strains of E. coli which produce only heat-stable enterotoxin in vitro. These latter pigs also developed diarrhea, but the tissue culture assay detects only LT and not heat-stable enterotoxin. It was concluded, therefore, that the diarrhea in the latter pigs was caused by heat-stable enterotoxin. These data support the hypothesis that in vivo production of LT, and probably heat-stable enterotoxin, occurs, and that both heat-labile and heat-stable types of E. coli enterotoxin are involved in the pathogenesis of diarrheal diseases caused by E. coli. Fluids collected from ligated jejunal loops of pigs at various times after exposure to several strains of enterotoxigenic E. coli were not found to contain E. coli enterotoxin by jejunal loop assays in pigs and rabbits or by exposure of adrenal cells in tissue culture to the fluids, with the exception of a single sample that was weakly positive in the tissue culture assay system. Pig loop fluids were found to be capable of inactivating preformed E. coli enterotoxin, even after the fluids and contents were heated to 100°C, which may explain the absence of demonstrable enterotoxin in ligated loop fluids. The data presented here suggest that the adrenal cell tissue culture system is a sensitive and specific assay system for the detection of LT in intestinal fluids and fecal materials.