Various techniques have been applied to the detection of skin reactivity associated with heat-labile Escherichia coli enterotoxin in fermenter-grown cultures of enterotoxigenic strains in syncase medium and in trypticase soy broth. Isolated products that were homogeneous, as determined by disc electrophoresis and sodium dodecyl sulfate gel electrophoresis, differed immunologically and in physicochemical characteristics depending on the strain and medium used, even though the products had similar specific activities in skin tests and in Chinese hamster ovary cells. In support of observations on porcine E.coli enterotoxin, and in contrast to the enterotoxin of Vibrio cholerae, the products were single polypeptide chains with molecular weights ranging from approximately 35,000 ot over 100,000 daltons. Proteolytic cleavage during culture and purification might account for some of the variations observed. The isolated products were almost 10(6)-fold less active than purified choleragen in causing morphologic alterations of Chinese hamster ovary cells, approximately 1,000-fold less active in skin tests, and at least 100-fold less active in rabbit ileal loops. In addition, only 1/100 as much active protein was produced by the strains employed as by V. cholerae. It is possible that accessory or host-derived factors are required. The most effective large-scale procedure for isolation from trypticase soy broth culture supernatants was a sequence of concentration by ultrafiltration, judicious (NH4)2SO4 precipitation, gel filtration on Sephadex G-150, and gel filtration on Agarose A5m, from which the E. coli products (like choleragen) are retarded in their elution. Toxin was isolated from an enterotoxigenic strain that caused diarrhea (total volume, 60-liters) for four days in a patient who had visited Mexico.